Transgenic bovine comprising human immunoglobulin loci and producing human immunoglobulin

ABSTRACT

The present invention relates to the production of a transgenic bovine which comprises a genetic modification that results in inactivation and loss of expression of its endogenous antibodies, and the expression of xenogenous antibodies, preferably human antibodies. This is effected by inactivation of the IgM heavy chain expression and, optionally, by inactivation of the Ig light chain expression, and by the further introduction of an artificial chromosome which results in the expression of non-bovine antibodies, preferably human antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.provisional patent application 60/311,625, filed Aug. 9, 2001 and U.S.provisional patent application 60/256,458, filed Dec. 20, 2000, and is acontinuation-in-part of U.S. utility application Ser. No. 09/714,185,filed Nov. 17, 2000, now abandoned which claims priority to U.S.provisional patent application 60/166,410, filed Nov. 19, 1999.

FIELD OF THE INVENTION

The present invention is a genetically modified ungulate that containseither part or all of a xenogenous antibody gene locus, which undergoesrearrangement and expresses a diverse population of antibody molecules.In particular, the xenogenous antibody gene may be of human origin. Inaddition, the present invention provides for an ungulate in whichexpression of the endogenous antibody genes is either reduced oreliminated. The genetic modifications in the ungulate (for example,bovine) are made using a combination of nuclear transfer and moleculartechniques. These cloned, transgenic ungulates provide a replenishable,theoretically infinite supply of xenogenous polyclonal antibodies,particularly human antibodies, which have use, e.g. as therapeutics,diagnostics and for purification purposes.

BACKGROUND OF THE INVENTION

In 1890, Shibasaburo Kitazato and Emil Behring reported an experimentwith extraordinary results; particularly, they demonstrated thatimmunity can be transferred from one animal to another by taking serumfrom an immune animal and injecting it into a non-immune one. Thislandmark experiment laid the foundation for the introduction of passiveimmunization into clinical practice. Today, the preparation and use ofhuman immunoglobulin (Ig) for passive immunization is standard medicalpractice. In the United States alone, there is a $1,400,000,000 perannum market for human Ig, and each year more than 16 metric tons ofhuman antibody is used for intravenous antibody therapy. Comparablelevels of consumption exist in the economies of most highlyindustrialized countries, and the demand can be expected to grow rapidlyin developing countries. Currently, human antibody for passiveimmunization is obtained from the pooled serum of human donors. Thismeans that there is an inherent limitation in the amount of humanantibody available for therapeutic and prophylactic usage. Already, thedemand exceeds the supply and severe shortfalls in availability havebeen routine.

In an effort to overcome some of the problems associated with theinadequate supply of human Ig, various technologies have been developed.For example, the production of human Ig by recombinant methods in tissueculture is routine. Particularly, the recombinant expression of human Igin CHO expression systems is well known, and is currently utilized forthe production of several human immunoglobulins (Igs) and chimericantibodies now in therapeutic use.

Mice retaining an unrearranged human immunoglobulin gene have beendeveloped for the production of human antibodies (e.g., monoclonalantibodies) (see, for example, WO98/24893; WO96/33735; WO 97/13852;WO98/24884; WO97/07671(EP 0843961); U.S. Pat. No. 5,877,397; U.S. Pat.No. 5,874,299; U.S. Pat. No. 5,814,318; U.S. Pat. No. 5,789,650; U.S.Pat. No. 5,770,429; U.S. Pat. No. 5,661,016; U.S. Pat. No. 5,633,425;U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,569,825; and U.S. Pat. No.5,545,806).

Additionally, WO00/10383 (EP 1106061) describes modifying a humanchromosome fragment and transferring the fragment into certain cells viamicrocell fusion.

Further, WO01/35735 describes a bovine IgM heavy chain knockout.

U.S. Pat. No. 5,849,992 issued Dec. 15, 1998 to Meade et al., as well asU.S. Pat. No. 5,827,690 issued Oct. 27, 1998 to Meade et al., describethe production of monoclonal antibodies in the milk of transgenicanimals including mice, sheep, pigs, cows, and goats wherein thetransgenic animals expressed human Ig genes under the control ofpromoters that provide for the expression of the antibodies in mammaryepithelial cells. Essentially, this results in the expression of theantibodies in the milk of such animals, for example a cow.

However, notwithstanding what has been previously reported, improvedmethods and enhanced transgenic animals, especially cows, that produceantibodies (particularly, polyclonal antibodies) of desired species,particularly human Igs, in the bloodstream and which produce an array ofdifferent antibodies which are specific to a desired antigen would behighly desirable. Most especially, the production of human Igs inungulates, such as cows, would be particularly beneficial given that (1)cows could produce large quantities of antibody, (2) cows could beimmunized with human or other pathogens and (3) cows could be used tomake human antibodies against human antigens. The availability of largequantities of polyclonal antibodies would be advantageous for treatmentand prophylaxis for infectious disease, modulation of the immune system,removal of human cells, such as cancer cells, and modulation of specifichuman molecules. While human Ig has been expressed in mice, it isunpredictable whether human Ig will be fractionally rearranged andexpressed in bovines, or other ungulates, because of differences inantibody gene structure, antibody production mechanism, and B cellfunction. In particular, unlike mice, cattle and sheep differ fromhumans in their immunophysiology (Lucier et al., J. Immunol. 161: 5438,1998; Pamg et al., J. Immunol. 157:5478, 1996; and Butler, Rev. Sci.Tech. 17:43, 2000). For example antibody gene diversification in bovinesand ovines relies much more on gene conversion than gene rearrangementas in humans and mice. Also, the primary location of B cells in humansand mice is in the bone marrow, whereas in bovines and ovines B cellsare located in the illeal Peyer's patch. Consequently, it would havebeen difficult, if not impossible, prior to the present invention, topredict whether immunoglobulin rearrangement and diversification of ahuman immunoglobulin loci would take place within the bovine (or otherungulate) B cell lineage. In addition, it would also have beenunpredictable whether a bovine would be able to survive, i.e., elicitits normal immune functions, in the absence of its endogenous Ig or withinterference from human antibodies. For example, it is not certain ifbovine B cells expressing human Ig would correctly migrate to the illealPeyer's Patch in bovines because this does not happen in humans. Also,it is not clear if human Fc receptor function; which mediates complementactivation, induction of cytokine release, and antigen removal; would benormal in a bovine system.

BRIEF DESCRIPTION OF THE INVENTION

It is an object of the invention to produce a transgenic ungulate (forexample, a transgenic bovine) that rearranges and expresses a human, orother species Ig gene locus. Preferably, this is accomplished by stablyintroducing a human chromosome fragment containing human Ig genes, inorder to produce a transgenic ungulate (for example, bovine) having Bcells that produce human or another species Ig, in addition to or inlieu of endogenous Igs. This may also be accomplished by integrating anucleic acid encoding a xenogenous immunoglobulin chain or xenogenousantibody into a chromosome of an ungulate. It is a further object of thepresent invention to produce transgenic ungulates (for example,transgenic bovines) wherein the expression of endogenous Ig has beenreduced or knocked out. For example, a nonsense or deletion mutation maybe introduced into a nucleic acid encoding an endogenous immunoglobulinchain or antibody.

It is a more specific object of the invention to produce a transgenicungulate (for example, a transgenic bovine) wherein the constant regionexon of the light chain loci and/or the mu constant regions exons havebeen knocked out, and an artificial chromosome containing a gene locusencoding another species' immunoglobulin, preferably human, has beenstably incorporated.

It is a more specific object of the invention to produce a clonedungulate (for example, a cloned bovine) by the use of nuclear transferand homologous recombination procedures wherein the endogenous constantregion exon of the light chain loci and/or the mu constant region exonsof the heavy chain locus have been knocked out, and an artificialchromosome(s) comprising xenogenous heavy and light chain Ig genes,preferably a human artificial chromosome(s) containing human heavy andlight chain Ig loci, has been stably introduced, resulting in atransgenic ungulate which produces Ig of another species, preferablyhuman, and which does not produce its endogenous Ig.

It is another object of the invention to produce an ungulate (forexample, a bovine) somatic or embryonic stem (ES) cell, preferably afibroblast or B cell, and more preferably a male somatic cell, whereinone or both alleles of the endogenous IgM heavy chain gene has beenmutated, for example, disrupted by homologous recombination. It is arelated object of the invention to produce a cloned ungulate (forexample, bovine) fetus and offspring wherein one or both alleles of theIgM heavy chain gene locus has been mutated, for example, disrupted byhomologous recombination.

It is still another object of the invention to produce an ungulate (forexample, a bovine) somatic or ES cell, preferably a fibroblast or Bcell, e.g., a female or male somatic cell, wherein one allele of the IgMheavy chain gene has been mutated, for example, disrupted by homologousrecombination.

It is a related object of the invention to produce a cloned (ungulate,for example, bovine) fetus or offspring wherein one allele of theendogenous heavy chain IgM gene has been mutated, for example, disruptedby homologous recombination.

It is still another object of the invention to produce male and femaleheavy and light chain hemizygous knockout (M and F Hemi H/L) fetuses andungulate calves by mating male and female ungulates (for example,bovines) which respectively contain a mutation, for example, adisruption of one allele of the endogenous IgM or a disruption of oneallele of an Ig light chain or by sequential homologous recombination.

It is still another object of the invention to produce a homozygousknockout (Homo H/L) fetus wherein both heavy chain alleles of the IgMgene have been disrupted and both alleles of the Ig light chain havebeen disrupted by sequential homologous recombination or by mating ofthe aforementioned male and female heavy and light chain hemizygousknockouts (M and F Hemi H/L).

It is another specific object of the invention to insert a nucleic acid(for example, an artificial chromosome) that contains genes necessaryfor the functional expression of non-ungulate Igs or their heavy orlight chains. Preferably, these Igs are human Igs produced byintroduction of nucleic acid encoding these Igs or Ig chains into a Homoor a Hemi H/L ungulate (for example, bovine) somatic cell, preferably afibroblast, and producing cloned ungulates (for example, cloned bovines)wherein the nucleic acid (for example, human artificial chromosome DNA)is transmitted into the germ line.

It is still another object of the invention to introduce an artificialchromosome, preferably a human artificial chromosome (HAC), thatcontains genes that provide for Ig expression into the aforementionedhomozygous knockout (Homo H/L) cells and generate ungulates (forexample, cattle) by nuclear transfer which express non-ungulate Igs,preferably human Igs, in response to immunization and which undergoaffinity maturation.

As used herein, by “artificial chromosome” is meant a mammalianchromosome or fragment thereof which has an artificial modification suchas the addition of a selectable marker, the addition of a cloning site,the deletion of one or more nucleotides, the substitution of one or morenucleotides, and the like. By “human artificial chromosome (HAC)” ismeant an artificial chromosome generated from one or more humanchromosome(s). An artificial chromosome can be maintained in the hostcell independently from the endogenous chromosomes of the host cell. Inthis case, the HAC can stably replicate and segregate along sideendogenous chromosomes. Alternatively, it may be translocated to, orinserted into, an endogenous chromosome of the host cell. Two or moreartificial chromosomes can be introduced to the host cell simultaneouslyor sequentially. For example, artificial chromosomes derived from humanchromosome #14 (comprising the Ig heavy chain gene), human chromosome #2(comprising the Ig kappa chain gene), and human chromosome #22(comprising the Ig lambda chain gene) can be introduced. Alternatively,an artificial chromosome(s) comprising both a xenogenous Ig heavy chaingene and Ig light chain gene, such as ΔHAC or ΔΔHAC, may be introduced.Preferably, the heavy chain loci and the light chain loci are ondifferent chromosome arms (i.e., on different side of the centromere).In still other preferred embodiments, the total size of the HAC is lessthan or equal to approximately 10, 9, 8, or 7 megabases.

It is still another object of the invention to provide a source of humanor other Ig for passive immunization derived from a transgenic ungulate(for example, a transgenic bovine) that contains and expresses Ig genescarried on an introduced nucleic acid (for example, an artificialchromosome, and preferably a human artificial chromosome (HAC))containing human Ig heavy and light chain genes. In the presentinvention, these nucleic acids (for example, HACs) include naturallyarranged segments of human chromosomes (human chromosomal fragments) orartificial chromosomes that comprise artificially engineered humanchromosome fragments, i.e., they may be rearranged relative to the humangenome.

It is yet another object of the invention to produce hybridomas andmonoclonal antibodies using B cells derived from the above-describedtransgenic ungulates (for example, transgenic bovines).

It is still another object of the invention to produce ungulateantiserum or milk that includes polyclonal human Ig. Such human Ig,preferably human IgG, may be used as intravenenous immunoglobulin (IVIG)for the treatment or prevention of disease in humans. The polyclonalhuman Ig are preferably reactive against an antigen of interest.

It is yet another object of the invention to produce a transgenicungulate with one or more mutations in an endogenous gene or genes. Thetransgenic ungulate is produced by inserting a cell, a chromatin massfrom a cell, or a nucleus from a cell into an oocyte. The cell has afirst mutation in an endogenous gene that is not naturally expressed bythe cell. The oocyte or an embryo formed from the oocyte is transferredinto the uterus of a host ungulate under conditions that allow theoocyte or the embryo to develop into a fetus. Preferably, the fetusdevelops into a viable offspring. In other preferred embodiments, thefirst mutation is introduced into the cell by inserting a nucleic acidcomprising a cassette which includes a promoter operably linked to anucleic acid encoding a selectable marker and operably linked to one ormore nucleic acids having substantial sequence identity to theendogenous gene to be mutated, whereby the cassette is integrated intoone endogenous allele of the gene. In other preferred embodiments, themutation is introduced in the cell by inserting into the cell a nucleicacid comprising a first cassette which includes a first promoteroperably linked to a nucleic acid encoding a first selectable marker andoperably linked to a first nucleic acid having substantial sequenceidentity to the endogenous gene to be mutated, whereby the firstcassette is integrated into a first endogenous allele of the geneproducing a first transgenic cell. Into the first transgenic cell isinserted a nucleic acid comprising a second cassette which includes asecond promoter operably linked to a nucleic acid encoding a secondselectable marker and operably linked to a second nucleic acid havingsubstantial sequence identity to the gene. The second selectable markerdiffers from the first selectable marker, and the second cassette isintegrated into a second endogenous allele of the gene producing asecond transgenic cell. In still other preferred embodiments, a cell isisolated from the embryo, the fetus, or an offspring produced from thefetus, and another mutation is introduced into a gene of the cell. Asecond round of nuclear transfer is then performed using the resultingcell, a chromatin mass from the cell, or a nucleus from the cell toproduce a transgenic ungulate with two or more mutations. The mutationsare in the same or different alleles of a gene or are in differentgenes. In preferred embodiments, the cell that is mutated is afibroblast (e.g., a fetal fibroblast). Preferably, the endogenous genethat is mutated is operably linked to an endogenous promoter that is notactive in a fibroblast. In other preferred embodiments, the endogenouspromoter operably linked to the endogenous gene that is mutated is lessthan 80, 70, 60, 50, 40, 30, 20, 10% as active as an endogenous promoteroperably linked to a endogenous housekeeping gene such as GAPDH.Promoter activity may be measured using any standard assay, such asassays that measure the level of mRNA or protein encoded by the gene(see, for example, Ausubel et al. Current Protocols in MolecularBiology, volume 2, p. 11.13.1–11.13.3, John Wiley & Sons, 1995). Thismethod for generating a transgenic ungulate has the advantage ofallowing a gene that is not expressed in the donor cell (i.e., the cellthat is the source of the genetic material used for nuclear transfer) tobe mutated.

Accordingly, the invention as claimed features a transgenic ungulatehaving one or more nucleic acids encoding all or part of a xenogenousimmunoglobulin (Ig) gene which undergoes rearrangement and expressesmore than one xenogenous Ig molecule. In a preferred embodiment, thenucleic acid encoding all or part of a xenogenous Ig gene issubstantially human. Preferably, the nucleic acid encodes an xenogenousantibody, such as a human antibody or a polyclonal antibody. In variousembodiments, the Ig chain or antibody is expressed in serum and/or milk.In other embodiments, the nucleic acid is contained within a chromosomefragment, such as a ΔHAC or a ΔΔHAC. In yet other embodiments, thenucleic acid is maintained in an ungulate cell independently from thehost chromosome.

In still other embodiments, the nucleic acid is integrated into achromosome of the ungulate. In yet other embodiments, the nucleic acidincludes unrearranged antibody light chain nucleic acid segments inwhich all of the nucleic acid segments encoding a V gene segment areseparated from all of the nucleic acid segments encoding a J genesegment by one or more nucleotides. In yet other embodiments, thenucleic acid includes unrearranged antibody heavy chain nucleic acidsegments in which either (i) all of the nucleic acid segments encoding aV gene segment are separated from all of the nucleic acid segmentsencoding a D gene segment by one or more nucleotides and/or (ii) all ofthe nucleic acid segments encoding a D gene segment are separated fromall of the nucleic acid segments encoding a J gene segment by one ormore nucleotides. In yet another preferred embodiment, the ungulate hasa mutation in one or both alleles of an endogenous Ig gene,alpha-(1,3)-galactosyltransferase gene, prion gene, and/or J chain gene.In other preferred embodiments, the ungulate has a nucleic acid encodingan exogenous J chain, such as a human J chain. Preferably, the ungulateis a bovine, ovine, porcine, or caprine.

In another aspect, the invention features a transgenic ungulate having amutation that reduces the expression of an endogenous antibody.Preferably, the mutation reduces the expression of functional IgM heavychain or substantially eliminates the expression of functional IgM heavychain. In other preferred embodiments, the mutation reduces theexpression of functional Ig light chain or substantially eliminates theexpression of functional Ig light chain. In yet other preferredembodiments, the mutation reduces the expression of functional IgM heavychain and functional Ig light chain, or the mutation substantiallyeliminates the expression of functional IgM heavy chain and functionalIg light chain. Preferably, the ungulate also has a mutation in one orboth alleles of an endogenous nucleic acid encodingalpha-(1,3)-galactosyltransferase, prion protein, and/or J chain. Inother preferred embodiments, the ungulate has a nucleic acid encoding anexogenous J chain, such as a human J chain. In another preferredembodiment, the ungulate has one or more nucleic acids encoding all orpart of a xenogenous Ig gene which undergoes rearrangement and expressesmore than one xenogenous Ig molecule. Preferably, the nucleic acidencoding all or part of a xenogenous Ig gene is substantially human. Inother preferred embodiments, the nucleic acid encodes a xenogenousantibody, such as a an antibody from another genus (e.g., a humanantibody) or a polyclonal antibody. In various embodiments, the Ig chainor antibody is expressed in serum. In other embodiments, the nucleicacid is contained within a chromosome fragment, such as a ΔHAC or aΔΔHAC. In yet other embodiments, the nucleic acid is maintained in anungulate cell independently from the host chromosome. In still otherembodiments, the nucleic acid is integrated into a chromosome of theungulate. Preferably, the ungulate is a bovine, ovine, porcine, orcaprine.

The invention also provides cells obtained from any of the ungulates ofthe invention or cells that are useful in the production of any of theungulates of the invention.

Accordingly, in another aspect, the invention features an ungulatesomatic cell having one or more nucleic acids encoding all or part of axenogenous Ig gene that is capable of undergoing rearrangement andexpressing one or more xenogenous Ig molecules in B cells. Preferably,the nucleic acid encoding all or part of a xenogenous Ig gene encodes axenogenous antibody. In various embodiments, the nucleic acid iscontained in a chromosome fragment, such as a ΔHAC or a ΔΔHAC. In yetother embodiments, the nucleic acid is maintained in an ungulate cellindependently from the host chromosome. In still other embodiments, thenucleic acid is integrated into a chromosome of the cell. In anotherembodiment, the nucleic acid is substantially human. Preferably, thexenogenous antibody is an antibody from another genus, such as a humanantibody. Preferably, the cell has a mutation in one or both alleles ofan endogenous nucleic acid encoding alpha-(1,3)-galactosyltransferase,prion protein, and/or J chain. In other preferred embodiments, the cellhas a nucleic acid encoding an exogenous J chain, such as a human Jchain. Exemplary ungulate cells include fetal fibroblasts and B-cells.Preferably, the ungulate is a bovine, ovine, porcine, or caprine.

In another aspect, the invention features an ungulate somatic cellhaving a mutation in a nucleic acid encoding an Ig heavy and/or lightchain. In preferred embodiments, the cell has a mutation in one or bothalleles of the IgM heavy chain or the Ig light chain. Exemplarymutations include nonsense and deletion mutations. Preferably, the cellhas a mutation in one or both alleles of an endogenous nucleic acidencoding alpha-(1,3)-galactosyltransferase, prion protein, and/or Jchain. In other preferred embodiments, the cell has a nucleic acidencoding an exogenous J chain, such as a human J chain. In preferredembodiments, the cell also has one or more nucleic acids encoding all orpart of a xenogenous Ig gene that is capable of undergoing rearrangementand expressing one or more xenogenous Ig molecules in B cells.Preferably, the nucleic acids encoding all or part of a xenogenous Iggene is substantially human and/or encodes a xenogenous antibody, suchas an antibody from another genus (e.g., a human antibody). In variousembodiments, the nucleic acid is contained in a chromosome fragment,whereby the nucleic acid is maintained in the ungulate cellindependently from the host chromosome. Preferred chromosome fragmentsinclude ΔHAC and ΔΔHAC. In yet other embodiments, the nucleic acid ismaintained in an ungulate cell independently from the host chromosome.In still other embodiments, the nucleic acid is integrated into achromosome of the cell. Exemplary ungulate cells include fetalfibroblasts and B-cells. Preferably, the ungulate is a bovine, ovine,porcine, or caprine.

In another aspect, the invention features a hybridoma formed from thefusion of an ungulate B-cell of the invention with a myeloma cell.Preferably, the hybridoma secretes an exogenous antibody, such as ahuman antibody.

The invention also provides methods for producing antibodies using anungulate of the invention. One such method involves administering one ormore antigens of interest to an ungulate having one or more nucleicacids encoding a xenogenous antibody gene locus. The nucleic acidsegments in the gene locus undergo rearrangement resulting in theproduction of antibodies specific for the antigen, and the antibodiesare recovered from the ungulate. In various embodiments, the nucleicacid encoding the xenogenous antibody gene locus is contained in achromosome fragment, such as a ΔHAC or a ΔΔHAC. Preferably, thechromosome fragment is maintained in an ungulate cell independently fromthe host chromosome. In other embodiments, the nucleic acid isintegrated into a chromosome of the ungulate. Preferably, the nucleicacid is substantially human. In other preferred embodiments, the lightchain of the antibodies and/or the heavy chain of the antibodies isencoded by a human nucleic acid. The antibodies may be monoclonal orpolyclonal. Monoclonal and polyclonal antibodies against particularantigen have a variety of uses; for example, they may be used asingredients in prophylactic or therapeutic compositions for infection ofpathogenic microorganisms such as bacteria or viruses. In variousembodiments, the antibodies are recovered from the serum or milk of theungulate. In preferred embodiments, the ungulate has a mutation thatreduces the expression of an endogenous antibody, that reduces theexpression of functional IgM heavy chain, or that reduces the expressionof functional Ig light chain. Preferably, the ungulate has a mutation inone or both alleles of an endogenous nucleic acid encodingalpha-(1,3)-galactosyltransferase, prion protein, and/or J chain. Inother preferred embodiments, the ungulate has a nucleic acid encoding anexogenous J chain, such as a human J chain. Preferably, the ungulate isa bovine, ovine, porcine, or caprine.

In a related aspect, the invention features another method of producingantibodies. This method involves recovering xenogenous antibodies froman ungulate having nucleic acid encoding a xenogenous antibody genelocus. The nucleic acid segments in the gene locus undergo rearrangementresulting in the production of xenogenous antibodies. In variousembodiments, the nucleic acid encoding a xenogenous antibody gene locusis contained in a chromosome fragment, such as a ΔHAC or a ΔΔHAC. Inparticular embodiments, the nucleic acid is maintained in an ungulatecell independently from the host chromosome. In still other embodiments,the nucleic acid is integrated into a chromosome of the ungulate.Preferably, the nucleic acid is substantially human. In particularembodiments, the light chain of the antibodies and/or the heavy chain ofthe antibodies is encoded by a human nucleic acid. The antibodies may bemonoclonal or polyclonal. In particular embodiments, polyclonalantibodies, such as IgG antibodies generated without immunization of theungulate with a specific antigen, are used as a therapeutic substitutefor IVIG (intraveneous immunoglobulin) produced from human serum. Invarious embodiments, the antibodies are recovered from the serum or milkof the ungulate. Preferably, the ungulate has a mutation that reducesthe expression of an endogenous antibody, reduces the expression offunctional IgM heavy chain, or reduces the expression of functional Iglight chain. Preferably, the ungulate has a mutation in one or bothalleles of an endogenous nucleic acid encodingalpha-(1,3)-galactosyltransferase, prion protein, and/or J chain. Inother preferred embodiments, the ungulate has a nucleic acid encoding anexogenous J chain, such as a human J chain. Preferably, the ungulate isa bovine, ovine, porcine, or caprine.

The invention also provides methods for producing transgenic ungulates.These methods may be used to produce transgenic ungulates having adesired mutation or having a desired xenogenous nucleic acid.

In one such aspect, the invention features a method of producing atransgenic ungulate that involves inserting a cell, a chromatin massfrom a cell, or a nucleus from a cell into an oocyte. The cell includesa first mutation in an endogenous antibody heavy chain and/or lightchain nucleic acid. The oocyte or an embryo formed from the oocyte istransferred into the uterus of a host ungulate under conditions thatallow the oocyte or the embryo to develop into a fetus. Preferably, thefetus develops into a viable offspring. Preferably, the cell used in theproduction of the transgenic ungulate has a mutation in one or bothalleles of an endogenous nucleic acid encodingalpha-(1,3)-galactosyltransferase, prion protein, and/or J chain. Inother preferred embodiments, the cell has a nucleic acid encoding anexogenous J chain, such as a human J chain. In other embodiments, thecell includes one or more nucleic acids encoding all or part of axenogenous Ig gene that is capable of undergoing rearrangement andexpressing one or more xenogenous Ig molecules in B cells. Preferably,the nucleic acid encoding all or part of a xenogenous Ig gene encodes axenogenous antibody. In yet other embodiments, the nucleic acid isintegrated into a chromosome of the cell. Preferably, the xenogenousantibody is an antibody from another genus, such as a human antibody. Inparticular embodiments, the nucleic acid is contained in a chromosomefragment, such as a ΔHAC or a ΔΔHAC. In other particular embodiments,the chromosome fragment is maintained in an ungulate cell independentlyfrom the host chromosome. Preferably, the ungulate is a bovine, ovine,porcine, or caprine.

In various embodiments of the above aspect, the method also includesisolating a cell from the embryo, the fetus, or an offspring producedfrom the fetus and introducing a second mutation in an endogenousantibody heavy chain and/or light chain nucleic acid in the cell. Thecell, a chromatin mass from the cell, or a nucleus from the cell isinserted into an oocyte, and the oocyte or an embryo formed from theoocyte is transferred into the uterus of a host ungulate underconditions that allow the oocyte or the embryo to develop into a fetus.

In other embodiments of the above aspect, the cell used for generationof the transgenic ungulate is prepared by a method that includesinserting into the cell a nucleic acid having a cassette which includesa promoter operably linked to a nucleic acid encoding a selectablemarker and operably linked to one or more nucleic acids havingsubstantial sequence identity to the antibody heavy chain or light chainnucleic acid. The cassette is integrated into one endogenous allele ofthe antibody heavy chain or light chain nucleic acid.

In other embodiments, the cell is produced by inserting into the cell anucleic acid having a first cassette which includes a first promoteroperably linked to a nucleic acid encoding a first selectable marker andoperably linked to a first nucleic acid having substantial sequenceidentity to the antibody heavy chain or light chain nucleic acid. Thefirst cassette is integrated into a first endogenous allele of theantibody heavy chain or light chain nucleic acid producing a firsttransgenic cell.

Into the first transgenic cell is inserted a nucleic acid having asecond cassette which includes a second promoter operably linked to anucleic acid encoding a second selectable marker and operably linked toa second nucleic acid having substantial sequence identity to theantibody heavy chain or light chain nucleic acid. The second selectablemarker differs from the first selectable marker. The second cassette isintegrated into a second endogenous allele of the antibody heavy chainor light chain nucleic acid producing a second transgenic cell.

In yet another aspect, the invention features another method ofproducing a transgenic ungulate. This method involves inserting a cellhaving one or more xenogenous nucleic acids into an oocyte. Thexenogenous nucleic acid encodes all or part of a xenogenous Ig gene, andthe gene is capable of undergoing rearrangement and expressing more thanone xenogenous Ig molecule in B cells. The oocyte or an embryo formedfrom the oocyte is transferred into the uterus of a host ungulate underconditions that allow the oocyte or the embryo to develop into a fetus.Preferably, the fetus develops into a viable offspring. Preferably, thenucleic acid encoding all or part of a xenogenous Ig gene encodes axenogenous antibody. In other preferred embodiments, the antibody is apolyclonal antibody. In yet other preferred embodiments, theimmunogloblulin chain or antibody is expressed in serum and/or milk. Invarious embodiments, the nucleic acid is contained in a chromosomefragment, such as a ΔHAC or a ΔΔHAC. The nucleic acid can be maintainedin an ungulate cell independently from the host chromosome or integratedinto a chromosome of the cell. Preferably, the nucleic acid issubstantially human. In other embodiments, the xenogenous antibody is anantibody from another genus, such as a human antibody. Preferably, theungulate is a bovine, ovine, porcine, or caprine.

In yet another related aspect, the invention features another method ofproducing a transgenic ungulate. This method involves inserting a cell,a chromatin mass from a cell, or a nucleus from a cell into an oocyte.The cell includes a first mutation in an endogenous gene that is notnaturally expressed by the cell. The oocyte or an embryo formed from theoocyte is transferred into the uterus of a host ungulate underconditions that allow the oocyte or the embryo to develop into a fetus.Preferably, the fetus develops into a viable offspring. Preferably, thegene that is mutated encodes an antibody,alpha-(1,3)-galactosyltransferase, prion protein, or J chain. In anotherpreferred embodiment, the cell used in the production of the transgenicungulate is a fibroblast, such as a fetal fibroblast.

In various embodiments of the above method, the cell is prepared byinserting into the cell a nucleic acid having a cassette which includesa promoter operably linked to a nucleic acid encoding a selectablemarker and operably linked to one or more nucleic acids havingsubstantial sequence identity to the gene; whereby the cassette isintegrated into one endogenous allele of the gene. In other embodiments,the cell is produced by inserting into the cell a nucleic acid having afirst cassette which includes a first promoter operably linked to anucleic acid encoding a first selectable marker and operably linked to afirst nucleic acid having substantial sequence identity to the gene. Thefirst cassette is integrated into a first endogenous allele of the geneproducing a first transgenic cell. Into the first transgenic cell isinserted a nucleic acid having a second cassette which includes a secondpromoter operably linked to a nucleic acid encoding a second selectablemarker and operably linked to a second nucleic acid having substantialsequence identity to the gene. The second selectable marker differs fromthe first selectable marker. The second cassette is integrated into asecond endogenous allele of the gene producing a second transgenic cell.

In other embodiments of the above aspect, the method also includesintroducing a second mutation into the transgenic ungulate. In theseembodiments, a cell is isolated from the embryo, the fetus, or anoffspring produced from the fetus, and a second mutation is introducedin an endogenous gene in the cell. The cell, a chromatin mass from thecell, or a nucleus from the cell is inserted into an oocyte, and theoocyte or an embryo formed from the oocyte is transferred into theuterus of a host ungulate under conditions that allow the oocyte or theembryo to develop into a fetus.

The ungulates of the invention can be used to produce antiserum or milkcontaining an antibody of interest. In one such aspect, the inventionfeatures ungulate antiserum having polyclonal human immunoglobulins.Preferably, the antiserum is from a bovine, ovine, porcine, or caprine.In another preferred embodiment, the Igs are directed against a desiredantigen.

In yet another aspect, the invention features ungulate milk havingpolyclonal human Igs. Preferably, the milk is from a bovine, ovine,porcine, or caprine. In another preferred embodiment, the Igs aredirected against a desired antigen.

In preferred embodiments of various aspects of the invention, the heavychain is a mu heavy chain, and the light chain is a lambda or kappalight chain. In other preferred embodiments, the nucleic acid encodingthe xenogenous immunoglobulin chain or antibody is in its unrearrangedform. Preferably, an antigen of interest is administered to a transgenicungulate having the xenogenous immunoglobulin gene locus and antibodiesreactive with the antigen of interest are produced. In other preferredembodiments, more than one class of xenogenous antibody is produced bythe ungulate. In various embodiments, more than one different xenogenousIg or antibody is produced by the ungulate. Preferred nuclear transfermethods include inserting a cell of the invention, a chromatin mass fromthe cell, or a nucleus from the cell into an enucleated or nucleatedoocyte, and transferring the oocyte or an embryo formed from the oocyteinto the uterus of a host ungulate under conditions that allow theoocyte or the embryo to develop into a fetus.

In other preferred embodiments of various aspects of the invention, theungulate has a mutation in one or both alleles of the endogenousalpha-(1,3)-galactosyltransferase, prion, and/or J chain nucleic acid.Preferably, the mutation reduces or eliminates the expression of theendogenous alpha-(1,3)-galactosyltransferase enzyme,galactosyl(α1,3)galactose epitope, prion protein, and/or J chain. Instill other preferred embodiments, the ungulate contains a xenogenous Jchain nucleic acid, such as a human J chain nucleic acid. Preferably,the ungulate produces human IgA or IgM molecules containing human Jchain. In various embodiments of the invention, the nucleic acid used tomutate an endogenous ungulate nucleic acid (e.g., a knockout cassettewhich includes a promoter operably linked to a nucleic acid encoding aselectable marker and operably linked to a nucleic acid havingsubstantial sequence identity to the gene to be mutated) is notcontained in a viral vector, such as an adenoviral vector or anadeno-associated viral vector. For example, the nucleic acid may becontained in a plasmid or artificial chromosome that is inserted into anungulate cell, using a standard method such as transfection orlipofection that does not involve viral infection of the cell. In yetanother embodiment, the nucleic acid used to mutate an endogenousungulate nucleic acid (e.g., a knockout cassette which includes apromoter operably linked to a nucleic acid encoding a selectable markerand operably linked to a nucleic acid having substantial sequenceidentity to the gene to be mutated) is contained in a viral vector, suchas an adenoviral vector or an adeno-associated viral vector. Accordingto this embodiment, a virus containing the viral vector is used toinfect an ungulate cell, resulting in the insertion of a portion or theentire viral vector into the ungulate cell.

Exemplary ungulates include members of the orders Perissodactyla andArtiodactyla, such as any member of the genus Bos. Other preferredungulates include sheep, big-horn sheep, goats, buffalos, antelopes,oxen, horses, donkeys, mule, deer, elk, caribou, water buffalo, camels,llama, alpaca, pigs, and elephants. Preferably, the transgenic ungulateexpresses an immunoglobulin chain or antibody from another genus, suchas an antibody from any other mammal.

As used herein, by “a nucleic acid in its pre-arranged or unrearrangedform” is meant a nucleic acid that has not undergone V(D)Jrecombination. In preferred embodiments, all of the nucleic acidsegments encoding a V gene segment of an antibody light chain areseparated from all of the nucleic acid segments encoding a J genesegment by one or more nucleotides. Preferably, all of the nucleic acidsegments encoding a V gene segment of an antibody heavy chain areseparated from all of the nucleic acid segments encoding a D genesegment by one or more nucleotides, and/or all of the nucleic acidsegments encoding a D gene segment of an antibody heavy chain areseparated from all of the nucleic acid segments encoding a J genesegment by one or more nucleotides. Preferably, a nucleic acid in itsunrearranged form is substantially human. In other preferredembodiments, the nucleic acid is at least 70, 80, 90, 95, or 99%identical to the corresponding region of a naturally-occurring nucleicacid from a human.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A contains an overview of the procedures used to produce a cowthat contains an Ig knockout and human artificial chromosome. The timeline in FIG. 1A is based on an estimated 18 months to prepare the Igknockout vector and generate knockout cells, 2 months to generatefetuses from the knockout cells, 9 months to perform subsequentknockouts, 9 months of gestation for calves to be born, 12 months beforeembryos can be produced from calves, and 6 months to perform the HACtransfers.

FIG. 1B contains an overview of the methods used to produce a cow thatcontains a mutation in an endogenous Ig gene and contains ΔHAC or ΔΔHAC.For the time line in FIG. 1B, it is estimated that 250 colonies arescreened per week for a total of 3,000 colonies in 3 months to isolatemale and female knockout cells. It is assumed that one or more knockoutcolonies are produced per 1,500 colonies. Homozygous knockout ungulatesmay be produced by (1) introducing a second Ig mutation in an isolatedknockout cell before nuclear transfer, (2) introducing a second Igmutation in a cell obtained from a embryo, fetus (e.g., fetus at ˜60gestation days), or offspring produced from a first round of nucleartransfer and using the resulting homozygous cell as the donor cell in asecond round of nuclear transfer, or (3) mating hemizygous ungulates. InFIGS. 1A and 1B, “Homo” denotes homozygous; “Hemi” denotes hemizygous;“H” denotes heavy chain; “L” denotes light chain; “HAC” denotes humanartificial chromosome; “HAC 1 ” denotes either HAC; and “HAC2 ” denotesa second HAC.

FIG. 2A contains a mu (IgM heavy chain) knockout construct according tothe invention. FIG. 2B is a restriction map of immunoglobulin loci froma Holstein cattle.

FIGS. 3A and 3B contain schematic illustrations of construct “pSTneoB”and “pLoxP-STneoB” that were used to produce the mu knockout DNAconstruct, which is illustrated in FIG. 3C. FIG. 3D is thepolynucleotide sequence of the 1.5 kb region of the genomic bovine muheavy chain locus that was used as the first region of homology in themu knockout construct (SEQ ID NO: 47). FIG. 3E is the polynucleotidesequence of the 3.1 kb region of the genomic bovine mu heavy chain locusthat was used as the second region of homology in the mu knockoutconstruct construct (SEQ ID NO: 48). In this sequence, each “n”represents any nucleotide or no nucleotide. The region of consecutive“n” nucleotides represents an approximately 0.9 to 1.0 kb region forwhich the polynucloetide sequence has not been determined. FIG. 3F is aschematic illustration of a puromycin resistant, bovine mu heavy chainknockout construct. FIG. 3G is the polynucleotide sequence of a bovinekappa light chain cDNA (SEQ ID NO: 60). All or part of this sequence maybe used in a kappa light chain knockout construct. Additionally, thiskappa light chain may be used to isolate a genomic kappa light chainsequence for use in a kappa light chain knockout construct.

FIG. 4 is a schematic illustration of the construction of ΔHAC andΔΔHAC.

FIG. 5 is a picture of an agarose gel showing the presence of genomicDNA encoding human heavy and light chains in ΔHAC fetuses.

FIG. 6 is a picture of an agarose gel showing the expression of humanCmu exons 3 and 4 in a ΔHAC fetus at 77 gestational days (fetus #5996).

FIG. 7 is a picture of an agarose gel showing the rearrangement ofendogenous bovine heavy chain in ΔHAC fetus #5996

FIG. 8 is a picture of an agarose gel showing the expression ofrearranged human heavy chain in ΔHAC fetus #5996.

FIG. 9 is a picture of an agarose gel showing the expression of thespliced constant region from the human heavy chain locus in ΔHAC fetus#5996.

FIG. 10 is a picture of an agarose gel showing the expression ofrearranged human heavy chain in ΔHAC fetus #5996.

FIG. 11A is the polynucleotide sequence of a rearranged human heavychain transcript from ΔHAC fetus #5996 (SEQ ID NO: 49). FIG. 11B is asequence alignment of a region of this sequence (“Query”) with a humananti-pneumococcal antibody (“Sbjct”) (SEQ ID NOs: 50 and 51,respectively). For the query sequence from ΔHAC fetus #5996, only thosenucleotides that differ from the corresponding nucleotides of the humananti-pneumococcal antibody sequence are shown.

FIGS. 12A and 12B are two additional polynucleotide sequences (SEQ IDNOs: 52 and 54) and their deduced amino acid sequences (SEQ ID NOs: 53and 55, respectively) of rearranged human heavy chain transcripts fromΔHAC fetus #5996.

FIG. 13 is a picture of an agarose gel demonstrating that ΔΔHAC fetus#5580 contains both human heavy and light chain immunoglobulin loci.

FIG. 14 is a picture of an agarose gel demonstrating that ΔΔHAC fetuses#5442A, and 5442B contain both human heavy and light chain loci.

FIG. 15 is a picture of an agarose gel showing the expression of thespliced mu constant region from the human heavy chain locus in ΔΔHACfetus #5542A.

FIG. 16 is a picture of an agarose gel showing the rearrangement andexpression of the human heavy chain locus in ΔΔHAC fetus #5868A.

FIG. 17 is a picture of an agarose gel showing rearrangement andexpression of the human Ig lambda locus in ΔΔHAC fetuses #5442A and5442B.

FIG. 18 is a picture of an agarose gel showing rearrangement andexpression of the human Ig lambda locus in ΔΔHAC fetus #5442A.

FIG. 19 is a picture of an agarose gel showing rearrangement andexpression of the human Ig lambda locus in ΔΔHAC fetus #5868A.

FIG. 20 is a polynucleotide sequence and the corresponded deduced aminoacid sequence of a rearranged human light chain transcript from ΔΔHACfetus #5442A (SEQ ID NOs: 56 and 57, respectively).

FIG. 21 is another polynucleotide sequence and the corresponding deducedamino acid sequence of a rearranged human light chain transcript fromΔΔHAC fetus #5442A (SEQ ID NOs: 58 and 59, respectively).

FIGS. 22A–22H are graphs of a FACS analysis of expression of humanlambda light chain and bovine heavy chain proteins by ΔΔHAC fetuses#5442A (FIGS. 22A–22D) and 5442B (FIGS. 22E–22H). Lymphocytes from thespleens of these fetuses were reacted with a phycoerytherin labeledanti-human lambda antibody (FIGS. 22C and 22D), a FITC labeledanti-bovine IgM antibody (FIGS. 22D and 22H), or no antibody (FIGS. 22A,22B, (22E, and 22F) and then analyzed on a FASCalibur cell sorter. Thepercent of cells that were labeled with one of the antibodies isdisplayed beneath each histogram.

FIG. 23 is a schematic illustration of the α-(1,3)-galactosyltransferaseknockout vector used to insert a puromycin resistance gene and atranscription termination sequence into the endogenousα-(1,3)-galactosyltransferase gene in bovine cells.

FIG. 24 is a schematic illustration of a BamHI-XhoI fragment containingexons 2, 3, and 4 that was used as a backbone for the AAV targetingvector. A neomycin resistance marker was used for insertionalmutagenesis of the locus by insertion into exon 4. The location of theannealing sites for the PCR primers that were used for subsequentconfirmation of appropriate targeting is indicated.

FIG. 25 is a schematic illustration of the construction of anadeno-associated viral construct designed to remove endogenous bovineIgH sequence.

FIG. 26 is a picture of an agarose gel showing the PCR analysis ofindividually transduced clones for appropriate targeting events. Thevector used in this experiment is shown in FIG. 24. PCR productsindicative of appropriate targeting are marked with asterisks.

FIG. 27 is table listing pregnancy rates for HAC carrying embryos.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, the present invention relates to the production of atransgenic ungulate, preferably a transgenic cow, wherein endogenous Igexpression has optionally been knocked out and a nucleic acid(preferably, an artificial chromosome) has been stably introduced thatcomprises genes which are necessary for the production of functionalantibodies of another species, preferably human. Thereby, a transgenicanimal may be obtained that does not produce its endogenous antibodies,but which instead produces antibodies of another species. Anynon-endogenous antibodies may be produced including, without limitation,human, non-human primate, dog, cat, mouse, rat, or guinea pigantibodies. While the production of human monoclonal antibodies in goatshas been previously reported, this has not been effected in cows, inserum or in any ungulate that do not express its endogenous antibodies.Furthermore, the insertion of germline (unrearragned) heavy or lightchain genes, the rearrangement of these genes and the expression ofdiversified antibody have not been performed in a transgenic ungulate.It is unpredictable whether such ungulates would survive because it isuncertain whether human Igs will be functionally expressed, or expressedin sufficient amounts to provide for adequate immune responses. Also, itis uncertain whether human chromosomes will be stably maintained intransgenic ungulates. Still further, it is uncertain whether thatungulate (for example, bovine) B cells will be able to express orproperly rearrange human or other non-endogenous Igs.

In a preferred embodiment of the present approach, xenogenousimmunoglobulin production is accomplished essentially by the combineduse of nuclear transfer, homologous recombination techniques, and theintroduction of artificial chromosomes carrying entire xenogenous Igloci. More specifically, the process preferably involves the targeteddisruption of one or both alleles of the IgM heavy chain gene, andoptionally one or both alleles of the Ig light chain gene, althoughxenogenous antibody production can also be accomplished in wild-typeanimals (i.e., animals without Ig knock outs). Gene knock outs may beeffected by sequential homologous recombination, then another matingprocedure. In a preferred embodiment, this is effected by initiallyeffecting targeted disruption of one allele of the IgM heavy chain geneof a male or female ungulate (for example, bovine) fetal fibroblast intissue culture using a suitable homologous recombination vector. The useof fetal fibroblasts is preferred over some other somatic cells as thesecells are readily propagated and genetically manipulated in tissueculture. However, the use of fetal fibroblasts is not essential to theinvention, and indeed other cell lines may be substituted therefor withequivalent results.

This process, of course, entails constructing a DNA construct havingregions of homology to the targeted IgM heavy chain allele such that theconstruct upon integration into an IgM heavy chain allele in theungulate genome disrupts the expression thereof. An exemplary vector forcarrying out such targeted disruption of an IgM allele is described inthe example which follows. In this regard, methods for constructingvectors that provide for homologous recombination at a targeted site arewell known to those skilled in the art. Moreover, in the presentinstance, the construction of a suitable vector is within the level ofskill in the art, given especially that the sequence of the bovine IgMheavy chain and Ig lambda light chain genes are known, as are thesequences of immunoglobulon genes from other ungulates (see below) Inorder to facilitate homologous recombination, the vectors used to effecthomologous recombination and inactivation of the IgM gene, respectively,comprise portions of DNA that exhibit substantial sequence identity tothe ungulate IgM heavy and Ig light chain genes. Preferably, thesesequences possessing at least 98% sequence identity, more preferably, atleast 99% sequence identity, and still more preferably will be isogenicwith the targeted gene loci to facilitate homologous recombination andtargeted deletion or inactivation.

Typically, and preferably the construct will comprise a marker gene thatprovides for selection of desired homologous recombinants, for example,fibroblast cells, wherein the IgM heavy chain gene and/or Ig light chaingene has been effectively disrupted. Exemplary marker genes includeantibiotic resistance markers, drug resistance markers, and greenfluorescent protein, among others. A preferred construct is shown inFIG. 2A and starting materials used to make this construct in FIGS. 3Aand 3B. Other constructs containing two regions of homology to anendogenous immunoglobulin gene, which flank a positive selection marker(e.g., an antibiotic resistance gene) that is operably linked to apromoter, may be generated using standard molecular biology techniquesand used in the methods of the present invention.

The mu knockout construct shown in FIGS. 2A and 3C was designed toremove the exons encoding the bovine immunoglobulin heavy chain constantregion, designated as “C-mu exons 1–4” and the two exons encoding thetransmembrane domain, designated “TM exons”.

To construct this vector, the region designated as “1”, an Xba1-Xho1fragment from the genomic mu heavy chain bovine sequence, was subclonedinto the commercial DNA vector, pBluescript (Stratagene, La Jolla,Calif.), previously cut with the enzymes XbaI and XhoI. Once thisfragment was cloned, there was a NotI restriction enzyme recognitionsequence adjacent to the Xbal site, used to insert a NotI fragment ofapproximately 3.5 Kb. This fragment contains a neomycin resistancemarker, described further below. If desired, other mu knock outconstructs may be constructed using the genomic mu heavy chain sequencefrom another ungulate breed, species, or genus (e.g., the mu heavy chainsequence deposited as Genbank accession number U63637 from a SwissBull/Holstein cross).

Once fragment “1” and the neomycin resistance marker were joinedtogether into pBluescript, there remained a Sac1 site adjacent to theneomycin resistance marker. The new construct was linearized with Sac1and converted to a blunt end by filling in the sticky ends left from theSac1 digest, using DNA polymerase.

The fragment designated “2” was isolated as an XhoI-BstI1071 fragmentand converted to a blunt-ended fragment by filling in the sticky endsleft from the Xho1 and BstI1071 enzymes, using DNA polymerase.

Once finished, the final construct contained region 2, the neomycinresistance marker and region 1, respectively.

For transfection of bovine fibroblasts, the construct was digested withthe restriction enzyme, Kpn1 (two Kpn1 sites are shown in the diagram)and the DNA fragment was used for homologous recombination.

The neomycin resistance construct was assembled as follows. A constructdesignated “pSTneoB” (Katoh et al., Cell Struct. Funct. 12:575, 1987;Japanese Collection of Research Biologicals (JCRB) deposit number:VE039) was designed to contain a neomycin resistance gene under thecontrol of an SV40 promoter and TK enhancer upstream of the codingregion. Downstream of the coding region is an SV40 terminator sequence.The neo cassette was excised from “pSTneoB” as a XhoI fragment. Afterthe ends of the fragment were converted to blunt ends using standardmolecular biology techniques, the blunt ended fragment was cloned intothe EcoRV site in the vector, pBS246 (Gibco/Life Technologies). Thissite is flanked by loxP sites. The new construct, designated“pLoxP-STNeoR”, was used to generate the mu knockout DNA construct. Thedesired fragment of this construct is flanked by loxP sites and NotIsites, which were originally present in the pBS246 cloning vector. Thedesired NotI fragment, which contains loxP-neo-loxP, was used forreplacement of the immunoglobulin mu constant region exons. The SV40promoter operably linked to the neomycin resistance gene activates thetranscription of the neomycin resistance gene, allowing cells in whichthe desired NotI fragment has replaced the mu constant region exons tobe selected based on their resulting antibiotic resistance.

After a cell line is obtained in which the IgM heavy chain allele hasbeen effectively disrupted, it is used as a nuclear transfer donor toproduce a cloned ungulate fetus (for example, a cloned bovine fetus) andeventually a fetus or animal wherein one of the IgM heavy alleles isdisrupted. Thereafter, a second round of gene targeted disruption can beeffected using somatic cells derived therefrom, e.g., fibroblasts, inorder to produce cells in which the second IgM heavy chain allele isinactivated, using a similar vector, but containing a differentselectable marker.

Preferably, concurrent to the first targeted gene disruption, a secondungulate (for example, bovine) somatic cell line is also geneticallymodified, which similarly may be of male or female origin. If the firstcell line manipulated is male, it is preferable to modify a female cellline; vice versa if the first cell line manipulated is female, it ispreferable to select a male cell line. Again, preferably, themanipulated cells comprise ungulate (for example, bovine) fetalfibroblasts.

In a preferred embodiment, the female fetal fibroblast is geneticallymodified so as to introduce a targeted disruption of one allele of theIg lambda light chain gene. This method similarly is carried out using avector having regions of homology to the ungulate (for example, bovine)Ig lambda light chain, and a selectable marker, which DNA construct isdesigned such that upon integration and homologous recombination withthe endogenous Ig light chain results in disruption (inactivation) ofthe targeted Ig lambda light gene.

Once a female fibroblast cell line is selected having the desiredtargeted disruption, it similarly is utilized as a donor cell fornuclear transfer or the DNA from such cell line is used as a donor fornuclear transfer.

Alternatively, this cell may be subjected to a second round ofhomologous recombination to inactivate the second Ig lambda light chainusing a similar DNA construct to that used to disrupt the first allele,but containing a different selectable marker.

As discussed in the background of the invention, methods for effectingnuclear transfer, and particularly for the production of cloned bovinesand cloned transgenic bovines have been reported and are described inU.S. Pat. No. 5,995,577 issued to Stice et al. and assigned toUniversity of Massachusetts. Still, alternatively the nuclear transfertechniques disclosed in WO 95/16670; WO 96/07732; WO 97/0669; or WO97/0668, (collectively, Roslin Methods) may be used. The Roslin methodsdiffer from the University of Massachusetts techniques in that they usequiescent rather than proliferating donor cells. All of these patentsare incorporated by reference herein in their entirety. These nucleartransfer procedures will produce a transgenic cloned fetus which can beused to produce a cloned transgenic bovine offspring, for example, anoffspring which comprises a targeted disruption of at least one alleleof the Ig light chain gene and/or IgM gene. After such cell lines havebeen created, they can be utilized to produce a male and female heavyand light chain hemizygous knockout (M and F Hemi H/L) fetus andoffspring. Moreover, these techniques are not limited to use for theproduction of transgenic bovines; the above techniques may be used fornuclear transfer of other ungulates as well.

Following nuclear transfer, production of desired animals may beaffected either by mating the ungulates or by secondary gene targetingusing the homologous targeting vector previously described.

As noted previously, a further object of the invention involves creatingmale and female heavy and light chain hemizygous knockouts wherein suchhemizygous knockouts are produced using the cell lines alreadydescribed. This may be affected either by mating of the offspringproduced according to the above described methods, wherein an offspringwhich comprises a disrupted allele of the IgM heavy chain gene is matedwith another offspring which comprises a disrupted allele of the Iglight chain. Alternatively, this may be affected by secondary genetargeting by manipulating a cell which is obtained from an offspringproduced according to the above-described procedures. This will compriseeffecting by homologous recombination targeted disruption of an alleleof the IgM heavy chain gene or allele of the Ig light chain. After acell line is produced which comprises a male and female heavy and lightchain hemizygous knockout (M and F Hemi H/L) it will be used to producea fetus or calf which comprises such a knockout. As noted, this iseffected either by mating or secondary gene targeting.

Once the male and female heavy and light chain hemizygous knockouts areobtained, cells from these animals may be utilized to create homozygousknockout (Homo H/L) fetuses. Again, this is affected either bysequential gene targeting or mating. Essentially, if affected by mating,this will involve mating the male heavy and light chain hemizygousknockout with a female heavy and light chain hemizygous knockout andselection of an offspring which comprises a homozygous knockout.Alternatively, the cells from the hemizygous knockout described abovemay be manipulated in tissue culture, so as to knock out the otherallele of the IgM or Ig light chain (lambda) gene. Secondary genetargeting may be preferred to mating as this may provide for more rapidresults, especially given that the gestation period of ungulates, suchas bovines, is relatively long.

Once homozygous knockouts (Homo H/L) have been obtained, they areutilized for introduction of a desired nucleic acid which contains genes(preferably, entire gene loci) for producing antibodies of a particularspecies, such as a human. Preferably, human artificial chromosomes areused, such as those disclosed in WO 97/07671 (EP 0843961) and WO00/10383(EP 1106061). These human artificial chromosomes also are described in acorresponding issued Japanese patent JP 30300092. Both of theseapplications are incorporated by reference in their entirety herein.Also, the construction of artificial human chromosomes that contain andexpress human immunoglobulin genes is disclosed in Shen et al., Hum.Mol. Genet. 6(8):1375–1382 (1997); Kuroiwa et al., Nature Biotechnol.18(10):1086–1090 (2000); and Loupert et al., Chromosome 107(4):255–259(1998), all of which are incorporated by reference in their entiretyherein. Human artificial chromosomes may also be utilized to introducexenogenous antibody genes into wild-type animal cells; this isaccomplished using the methods described above. Introduction ofartificial chromosome into animal cells, especially fetal fibroblastcells can be performed by microcell fusion as described herein.

In an alternative to the use of human artificial chromosome, nucleicacid encoding immunoglobulin genes may be integrated into the chromosomeusing a YAC vector, BAC vector, or cosmid vector. Vectors comprisingxenogenous Ig genes (WO98/24893, WO96/33735, WO 97/13852, WO98/24884)can be introduced to fetal fibroblasts cells using known methods, suchas electroporation, lipofection, fusion with a yeast spheroplastcomprising a YAC vector, and the like. Further, vectors comprisingxenogenous Ig genes can be targeted to the endogenous Ig gene loci ofthe fetal fibroblast cells, resulting in the simultaneous introductionof the xenogenous Ig gene and the disruption of the endogenous Ig gene.

Integration of a nucleic acid encoding a xenogenous immunoglobulin genemay also be carried out as described in the patents by Lonberg et al.(supra). In the “knockin” construct used for the insertion of xenogenousimmunoglobulin genes into a chromosome of a host ungulate, one or moreimmunoglobulin genes and an antibiotic resistance gene may beoperably-linked to a promoter which is active in the cell typetransfected with the construct. For example, a constitutively active,inducible, or tissue specific promoter may be used to activatetranscription of the integrated antibiotic resistance gene, allowingtransfected cells to be selected based on their resulting antibioticresistance. Alternatively, a knockin construct in which the knockincassette containing the Ig gene(s) and the antibiotic resistance gene isnot operably-linked to a promoter may be used. In this case, cells inwhich the knockin cassette integrates downstream of an endogenouspromoter may be selected based on the resulting expression of theantibiotic resistance marker under the control of the endogenouspromoter. These selected cells may be used in the nuclear transferprocedures described herein to generate a transgenic ungulate containinga xenogenous immunoglobulin gene integrated into a host chromosome.

Using similar methodologies, it is possible to produce and insertartificial chromosomes containing genes for expression of Ig ofdifferent species such as dog, cat, other ungulates, non-human primatesamong other species. As discussed above, and as known in the art,immunoglobulin genes of different species are well known to exhibitsubstantial sequence homology across different species.

Once it has been determined that the inserted artificial chromosome, forexample, a human artificial chromosome, has been stably introduced intoa cell line, e.g., a bovine fetal fibroblast, it is utilized as a donorfor nuclear transfer. This may be determined by PCR methods. Similarly,animals are obtained which comprise the homozygous knockout, and furthercomprise a stably introduced nucleic acid, such as a human artificialchromosome. After calves have been obtained which include the stablyincorporated nucleic acid (for example, human artificial chromosome),the animals are tested to determine whether they express human Ig genesin response to immunization and affinity maturation.

Modifications of the overall procedure described above may also beperformed. For example, xenogenous Ig genes may be introduced first andthan endogenous Ig genes may be inactivated. Further, an animalretaining xenogenous Ig genes may be mated with an animal in which anendogenous Ig gene is inactivated. While the approaches to be utilizedin the invention have been described above, the techniques that areutilized are described in greater detail below. These examples areprovided to illustrate the invention, and should not be construed aslimiting. In particular, while these examples focus on transgenicbovines, the methods described may be used to produce and test anytransgenic ungulate.

Knockout Procedures to Produce Transgenic Ungulates that Express HumanIgs

As discussed above, the present invention relates to the production ofHomo H/L fetuses or calves. The approach is summarized in FIG. 1. Thereare three schemes outlined therein. The first relies on successiveknockouts in regenerated fetal cell lines. This approach is thetechnically most difficult and has the highest level of risk but asnoted above potentially yields faster results than breeding approaches.The other two schemes rely on breeding animals. In the second scheme,only single knockouts of heavy and light chain genes are required inmale and female cell lines, respectively. This scheme does not rely onregeneration of cell lines and is technically the simplest approach buttakes the longest for completion. Scheme 3 is an intermediate betweenschemes 1 and 2. In all schemes only Homo H/L fetuses are generatedbecause of potential difficulties in survival and maintenance of HomoH/L knockout calves. If necessary, passive immunotherapy can be used toincrease the survival of Homo H/L knockout calves.

Experimental Design The present invention preferably involves theproduction of a hemizygous male heavy chain knockout (M Hemi H) and ahemizygous female light chain knockout (F Hemi L) and the production of40 day fetuses from these targeted deletions. The cells from the embryosare harvested, and one allele of the light locus is targeted in the MHemi H cells and one allele of the heavy chain locus is targeted in theF Hemi L cells resulting in cells with hemizygous deletions of both theH and L loci (Hemi H/L). These cells are used to derive 40 day fetusesfrom which fibroblasts are isolated.

The M Hemi H/L fibroblasts are targeted with the other H chain allele tocreate M Homo H/Hemi L, and the F Hemi H/L are targeted with the other Lchain allele to create F Homo L/Hemi H. In order to create homozygousdeletions, higher drug concentrations are used to drive homozygoustargeting. However, it is possible that this approach may not besuccessful and that breeding may be necessary. An exemplary strategywhich relies on cre/lox targeting of the selection cassette allows thesame selective systems to be used for more than one targeted deletion.These fibroblasts are cloned and 40 day fetuses harvested and fibroblastcells isolated. The fetal cells from this cloning are targeted toproduce homozygous deletions of either the H or L loci resulting in MHomo H/L and F Homo H/L fetal fibroblasts. These fibroblasts are clonedand 40 day fetuses derived and fibroblasts isolated. The Homo H/L fetalfibroblasts are then used for incorporation of the HAC optionally by theuse of breeding procedures.

Library Construction Fetal fibroblast cells are used to construct agenomic library. Although it is reported to be significant that thetargeting construct be isogenic with the cells used for cloning, it isnot essential to the invention. For example, isogenic, substantiallyisogenic, or nonisogenic constructs may be used to produce a mutation inan endogenous immunoglobulin gene. In one possible method, Holsteincattle, which genetically contain a high level of inbreeding compared toother cattle breeds, are used. We have not detected any polymorphisms inimmunoglobulin genes among different animals. This suggests thatsequence homology should be high and that targeting with nonisogenicconstructs should be successful.

A library is constructed from one male cell line and one female cellline at the same time that the “clonability” testing is being conducted.It is envisioned that at the end of the process, a library will beproduced and a number of different fetal cell lines will be tested andone cell line chosen as the best for cloning purposes.

Genomic libraries are constructed using high molecular weight DNAisolated from the fetal fibroblast cells. DNA is size fractionated andhigh molecular weight DNA between 20–23 Kb is inserted into the lambdaphage vector LambdaZap or LambdaFix. The inventors have had excellentsuccess with Stratagene prepared libraries. Therefore, DNA is isolatedand the size selected DNA is sent to Stratagene for library preparation.To isolate clones containing bovine heavy and light chains, radiolabeledIgM cDNA and radiolabeled light chain cDNA is used. Additionally, lightchain genomic clones are isolated in case it is necessary to delete thelocus. Each fetal cell library is screened for bovine heavy and lightchain containing clones. It is anticipated that screening approximately10⁵–10⁶ plaques should lead to the isolation of clones containing eitherthe heavy chain or light chain locus. Once isolated, both loci aresubcloned into pBluescript and restriction mapped. A restriction map ofthese loci in Holsteins is provided in FIG. 2B (Knight et al. J Immunol140(10):3654–9, 1988). Additionally, a map from the clones obtained ismade and used to assemble the targeting construct.

Production of Targeting Constructs Once the heavy and light chain genesare isolated, constructs are made. The IgM construct is made by deletingthe IgM constant region membrane domain. As shown by Rajewsky andcolleagues in mice, deletion of the membrane domain of IgM results in ablock in B cell development since surface IgM is a required signal forcontinued B cell development (Kitamura et al., Nature 350:423–6). Thushomozygous IgM cattle lack B cells. This should not pose a problem sincein the present strategy no live births of animals lacking functional Igare necessary. However, if necessary, passive immunotherapy may be usedto improve the survival of the animals until the last step when thehuman Ig loci are introduced.

An exemplary targeting construct used to effect knockout of the IgMheavy chain allele is shown below in FIG. 2A. For the heavy chain, themembrane IgM domain is replaced with a neomycin cassette flanked by loxP sites. The attached membrane domain is spliced together with the neocassette such that the membrane domain has a TAG stop codon insertedimmediately 5′ to the lox P site ensuring that the membrane domain isinactivated. This is placed at the 5′ end of the targeting constructwith approximately 5–6 kilobases of 3′ chromosomal DNA.

If increasing drug concentrations does not allow deletion of the secondallele of either IgM heavy or light chains, the cre/lox system (reviewedin Sauer, 1998, Methods 14:381–392) is used to delete the selectablemarker. As described below, the cre/lox system allows the targeteddeletion of the selectable marker. All selectable markers are flankedwith loxP sequences to facilitate deletion of these markers if thisshould be necessary.

The light chain construct contains the bovine lambda chain constantregion (e.g., the lambda light chain constant region found in Genbankaccession number AF396698 or any other ungulate lambda light chainconstant region) and a puromycin resistance gene cassette flanked by loxP sites and will replace the bovine gene with a puromycin cassetteflanked by lox P sites. Approximately 5–6 kilobases of DNA 3′ to thelambda constant region gene will be replaced 3′ to the puromycinresistance gene. The puromycin resistance gene will carry lox P sites atboth 5′ and 3′ ends to allow for deletion if necessary. Due to the highdegree of homology between ungulate antibody genes, the bovine lambdalight chain sequence in Genbank accession number AF396698 is expected tohybridize to the genomic lambda light chain sequence from a variety ofungulates and thus may be used in standard methods to isolate variousungulate lambda light chain genomic sequences. These genomic sequencesmay be used in standard methods, such as those described herein, togenerate knockout constructs to inactivate endogenous lambda lightchains in any ungulate.

A kappa light chain knockout construct may be constructed similarlyusing the bovine kappa light chain sequence in FIG. 3G or any otherungulate kappa light chain sequence. This bovine kappa light chain maybe used as a hybridization probe to isolate genomic kappa light chainsequences from a variety of ungulates. These genomic sequences may beused in standard methods, such as those described herein, to generateknockout constructs to inactivate endogenous kappa light chains in anyungulate.

Additional ungulate genes may be optionally mutated or inactivated. Forexample, the endogenous ungulate Ig J chain gene may be knocked out toprevent the potential antigenicity of the ungulate Ig J chain in theantibodies of the invention that are administered to humans. For theconstruction of the targeting vector, the cDNA sequence of the bovine IgJ chain region found in Genbank accession number U02301 may be used.This cDNA sequence may be used as a probe to isolate the genomicsequence of bovine Ig J chain from a BAC library such as RPC1-42 (BACPACin Oakland, Calif.) or to isolate the genomic sequence of the J chainfrom any other ungulate. Additionally, the human J chain coding sequencemay be introduced into the ungulates of present invention for thefunctional expression of human IgA and IgM molecules. The cDNA sequenceof human J chain is available from Genbank accession numbers AH002836,M12759, and M12378. This sequence may be inserted into an ungulate fetalfibroblast using standard methods, such as those described herein. Forexample, the human J chain nucleic acid in a HAC, YAC vector, BACvector, cosmid vector, or knockin construct may be integrated into anendogenous ungulate chromosome or maintained independently of endogenousungulate chromosomes. The resulting transgenic ungulate cells may beused in the nuclear transfer methods described herein to generate thedesired ungulates that have a mutation that reduces or eliminates theexpression of functional ungulate J chain and that contain a xenogenousnucleic acid that expresses human J chain.

Additionally, the ungulate α-(1,3)-galactosyltransferase gene may bemutated to reduce or eliminate expression of thegalactosyl(α1,3)galactose epitope that is produced by theα-(1,3)-galactosyltransferase enzyme. If human antibodies produced bythe ungulates of the present invention are modified by this carbohydrateepitope, these glycosylated antibodies may be inactivated or eliminated,when administered as therapeutics to humans, by antibodies in therecipients that are reactive with the carbohydrate epitope. To eliminatethis possible immune response to the carbohydrate epitope, the sequenceof bovine alpha-(1,3)-galactosyltransferase gene may be used to design aknockout construct to inactive this gene in ungulates (Genbank accessionnumber J04989; Joziasse et al., J. Biol. Chem. 264(24):14290–7, 1989).This bovine sequence or the procine alpha-(1,3)-galactosyltransferasesequence disclosed in U.S. Pat. Nos. 6,153,428 and 5,821,117 may be usedto obtain the genomic alpha-(1,3)-galactosyltransferase sequence from avariety of ungulates to generate other ungulates with reduced oreliminated expression of the galactosyl(α1,3)galactose epitope.

If desired, the ungulate prion gene may be mutated or inactivated toreduce the potential risk of an infection such as bovine spongiformencephalopathy (BSE). For the construction of the targeting vector, thegenomic DNA sequence of the bovine prion gene may be used (Genbankaccession number AJ298878). Alternatively, this genomic prion sequencemay be used to isolate the genomic prion sequence from other ungulates.The prior gene may be inactivated using standard methods, such as thosedescribed herein or those suggested for knocking out thealpha-(1,3)-galactosyltransferase gene or prion gene in sheep (Denninget al., Nature Biothech., 19: 559–562, 2001).

For targeting the second allele of each locus, it may be necessary toassemble a new targeting construct containing a different selectablemarker, if the first selectable marker remains in the cell. As describedin Table 1, a variety of selection strategies are available and may becompared and the appropriate selection system chosen. Initially, thesecond allele is targeted by raising the drug concentration (forexample, by doubling the drug concentration). If that is not successful,a new targeting construct may be employed.

The additional mutations or the gene inactivation mentioned above may beincorporated into the ungulates of the present invention using variousmethodologies. Once a transgenic ungulate cell line is generated foreach desired mutation, crossbreeding may be used to incorporate theseadditional mutations into the ungulates of the present invention.Alternatively, fetal fibroblast cells which have these additionalmutations can be used as the starting material for the knockout ofendogenous Ig genes and/or the introduction of xenogenous Ig genes.Also, fetal fibroblast cells having a knockout mutation in endogenous Iggenes and/or containg xenogenous Ig genes can be uses as a startingmaterial for these additional mutations or inactivations.

Targeted Deletion of Ig Loci Targeting constructs are introduced intoembryonic fibroblasts, e.g., by electroporation. The cells whichincorporate the targeting vector are selected by the use of theappropriate antibiotic. Clones that are resistant to the drug of choicewill be selected for growth. These clones are then subjected to negativeselection with gancyclovir, which will select those clones which haveintegrated appropriately. Alternatively, clones that survive the drugselection are selected by PCR. It is estimated that it will be necessaryto screen at least 500–1000 clones to find an appropriately targetedclone. The inventors' estimation is based on Kitamura (Kitamura et al.,Nature 350:423–6, 1991) who found that when targeting the membranedomain of IgM heavy chain constant region approximately 1 in 300 neoresistant clones were properly targeted. Thus, it is proposed to poolclones into groups of 10 clones in a 96 well plate and screen pools of10 clones for the targeted clones of choice. Once a positive isidentified, single clones isolated from the pooled clone will bescreened. This strategy should enable identification of the targetedclone.

Because fibroblasts move in culture it is difficult to distinguishindividual clones when more than approximately ten clones are producedper dish. Further, strategies may be developed for clonal propagationwith high efficiency transfection. Several reasonable strategies, suchas dilution cloning, may be used.

Cre/Lox Excision of the Drug Resistance Marker As shown above, exemplarytargeting constructs contain selectable markers flanked by loxP sites tofacilitate the efficient deletion of the marker using the cre/loxsystem. Fetal fibroblasts carrying the targeting vector are transfectedvia electroporation with a Cre containing plasmid. A recently describedCre plasmid that contains a GFPcre fusion gene [Gagneten S. et al.,Nucleic Acids Res 25:3326–31 (1997)] maybe used. This allows the rapidselection of all clones that contain Cre protein. These cells areselected either by FACS sorting or by manual harvesting of greenfluorescing cells via micromanipulation. Cells that are green areexpected to carry actively transcribed Cre recombinase and hence deletethe drug resistance marker. Cells selected for Cre expression are clonedand clones analyzed for the deletion of the drug resistance marker viaPCR analysis. Those cells that are determined to have undergone excisionare grown to small clones, split and one aliquot is tested in selectivemedium to ascertain with certainty that the drug resistance gene hasbeen deleted. The other aliquot is used for the next round of targeteddeletion.

TABLE 1 Selectable markers and drugs for selection Gene Drug Neo^(r)G418¹ Hph Hygromycin B² Puro Puromycin³ Ecogpt Mycophenolic acid⁴ BsrBlasticidin S⁵ HisD Histidinol⁶ DT-A Diphtheria toxin⁷ ¹Southern PJ,Berg P. 1982. Transformation of mammalian cells to antibiotic resistancewith a bacterial gene under control of the SV40 early region promoter. JMol AppI Genet 1:327–41. ²Santerre RF, Allen NE, Hobbs JN Jr, Rao RN,Schmidt RJ. 1984. Expression of prokaryotic genes for hygromycin B andG418 resistance as dominant-selection markers in mouse L cells. Gene30:147–56. ³Wirth M, Bode J, Zettlmeissl G, Hauser H. 1988. Isolation ofoverproducing recombinant mammalian cell lines by a fast and simpleselection procedure. Gene 73:419–26. ⁴Drews RE, Kolker MT, Sachar DS,Moran GP, Schnipper LE. 1996. Passage to nonselective media transientlyalters growth of mycophenolic acid-resistant mammalian cells expressingthe escherichia coli xanthine-guanine phosphoribosyltransferase gene:implications for sequential selection strategies. Anal Biochem235:215–26. ⁵Karreman C. 1998. New positive/negative selectable markersfor mammalian cells on the basis of Blasticidin deaminase-thymidinekinase fusions. Nucleic Acids Res 26:2508–10. ⁶Hartman SC, Mulligan RG.1988. Two dominant-acting selectable markers for gene transfer studiesin mammalian cells. Proc Natl Acad Sci U.S.A. 85:8047–51. ⁷Yagi T, NadaS., Watanabe N, Tamemoto H, Kohmura N, Ikawa Y, Aizawa S. 1993. A novelnegative selection for homologous recombinants using diphtheria toxin Afragment gene. Anal Biochem 214:77–86.Application of Targeting Strategies to Altering Immunoglobulin Genes ofOther Ungulates

To alter immunoglobulin genes of other ungulates, targeting vectors aredesigned to contain three main regions. The first region is homologousto the locus to be targeted. The second region is a drug selectionmarker that specifically replaces a portion of the targeted locus. Thethird region, like the first region, is homologous to the targeted locusbut is not contiguous with the first region in the wild type genome.Homologous recombination between the targeting vector and the desiredwild type locus results in deletion of locus sequences between the tworegions of homology represented in the targeting vector and replacementof that sequence with a drug resistance marker. In preferredembodiments, the total size of the two regions of homology isapproximately 6 kilobases, and the size of the second region thatreplaces a portion of the targeted locus is approximately 2 kilobases.This targeting strategy is broadly useful for a wide range of speciesfrom prokaryotic cells to human cells. The uniqueness of each vectorused is in the locus chosen for gene targeting procedures and thesequences employed in that strategy. This approach may be used in allungulates, including, without limitation, goats (Capra hircus), sheep(Ovis aries), and the pig (Sus scrufa), as well as cattle (Bos taurus).

The use of electroporation for targeting specific genes in the cells ofungulates may also be broadly used in ungulates. The general proceduredescribed herein is adaptable to the introduction of targeted mutationsinto the genomes of other ungulates. Modification of electroporationconditions (voltage and capacitance) may be employed to optimize thenumber of transfectants obtained from other ungulates.

In addition, the strategy used herein to target the heavy chain locus incattle (i.e., removal of all coding exons and intervening sequencesusing a vector containing regions homologous to the regions immediatelyflanking the removed exons) may also be used equally well in otherungulates. For example, extensive sequence analysis has been performedon the immunoglobulin heavy chain locus of sheep (Ovis aries), and thesheep locus is highly similar to the bovine locus in both structure andsequence (Genbank accession numbers Z71572, Z49180 through Z49188,M60441, M60440, AF172659 through AF172703). In addition to the largenumber of cDNA sequences reported for rearranged Ovis ariesimmunoglobulin chains, genomic sequence information has been reportedfor the heavy chain locus, including the heavy chain 5′ enhancer(Genbank accession number Z98207), the 3′ mu switch region (Z98680) andthe 5′ mu switch region (Z98681). The complete mRNA sequence for thesheep secreted form of the heavy chain has been deposited as accessionnumber X59994. This deposit contains the entire sequence of four codingexons, which are very homologous to the corresponding bovine sequence.

Information on the sheep locus was obtained from Genbank and used todetermine areas of high homology with bovine sequence for the design ofprimers used for PCR analysis. Because non-isogenic DNA was used totarget bovine cells, finding areas of high homology with sheep sequencewas used as an indicator that similar conservation of sequences betweenbreeds of cow was likely. Given the similarity between the sequences andstructures of the bovine and ovine immunoglobulin loci, it would beexpected that the targeting strategies used to remove bovineimmunoglobulin loci could be successfully applied to the ovine system.In addition, existing information on the pig (Sus scrofa, accessionnumber S42881) and the goat (Capra hircus, accession number AF140603),indicates that the immunoglobulin loci of both of these species are alsosufficiently similar to the bovine loci to utilize the present targetingstrategies.

Procedures for Insertion of HACs

Essentially, male and female bovine fetal fibroblast cell linescontaining human artificial chromosome sequences (#14fg., #2fg., and#22fg.) are obtained and selected and used to produce cloned calves fromthese lines.

For example, HACs derived from human chromosome #14 (“#14fg,” comprisingthe Ig heavy chain gene), human chromosome #2 (“#2fg,” comprising the Igkappa chain gene) and human chromosome #22 (“#22fg,” comprising the Iglambda chain gene) can be introduced simultaneously or successively.

The transmission of these chromosome fragments is tested by mating amale #14fg. animal to female #2fg. and #22fg. animals and evaluatingoffspring. If transmission is successful then the two lines are mated toproduce a line containing all three chromosome fragments.

Also, #14fg., #2fg., and #22fg. chromosome fragments may be insertedinto Homo H/L fetal cells and used to generate cloned calves or crosstransgenic HAC calves with Homo H/L calves. Alternatively, other HACs,such as ΔHAC or ΔΔHAC, may be introduced as described in Example 2 orintroduced using any other chromosome transfer method.

Rationale Germline transmission of HACs should be useful for introducingthe HACs into the Ig knockout animals and in propagating animals inproduction herds. The concern in propagation of HACs through thegermline is incomplete pairing of chromosomal material during meiosis.However, germline transmission has been successful in mice as shown byTomizuka et al. (Proc. Natl. Acad. Sci. USA, 97:722, 2000).

The strategy outlined in FIG. 1A consists of inserting #14fg. into amale line of cells and #2fg. and #22fg. each into female cell lines.Calves retaining a HAC are produced and germline transmission can betested both through females and males. Part of the resulting offspring(˜25%) should contain both heavy and light chain HACs. Further crossingshould result in a line of calves containing all three chromosomalfragments. These animals are used for crossing with Homo H/L animals,produced from fetal cells as previously described.

Experimental Design Cells are obtained from the original screening ofcell lines. These may be Holstein or different lines than those usedabove. This allows crossing while maintaining as much genetic variationin the herd as possible. Introduction of HACs into cell lines andselection of positive cell lines is then effected. Selected cell linesare used for nuclear transfer and calves are produced. Starting at 12months of age semen and eggs are collected, fertilized, and transferredinto recipient animals. Cell samples are taken for DNA marker analysisand karyotyping. Beginning at birth, blood samples are taken andanalyzed for the presence of human Ig proteins.

As indicated above, HACs are also transferred into Homo H/L cell linesusing the procedures developed in the above experiments.

Testing for Human Ig Expression

The goal of the experiment is to generate male Homo H cells and clonedfetuses, to insert one or more HACs that together contain human IgH andhuman IgL loci (such as HAC #14fg. and #22fg.) into Homo H cells andgenerate calves, and to test expression of human Ig response toimmunization and affinity maturation. This is carried out as follows.

Experimental Design Homo H cells are generated from Hemi H cellsproduced as described previously. The double knockout is produced eitherby antibiotic selection or a second insertion. HACs are transferred intothese cells as described previously. Calves are produced by nucleartransfer. Testing calves retaining a HAC begins shortly after birth andincludes (1) evaluation for human Ig expression, (2) response toimmunization, (3) affinity maturation, and (4) transmission of the HACsto offspring.

Human Ig expression is monitored by bleeding the animals and assayingfor the presence of human heavy and light chain expression by ELISA,RT-PCR, or FACS analysis. Once it has been determined that the animalsproduce human Ig, animals are immunized with tetanus toxoid in adjuvant.Animals are bled once a week following immunization and responses toantigen determined via ELISA or FACS and compared to pre-bleedscollected before immunization. One month after the initial immunization,animals are boosted with an aqueous form of the antigen. One weekfollowing the boost, the animals are bled and response to antigenmeasured via ELISA or FACS and compared to the prebleed. The ELISA orFACS assay permits measurement of most of the titer of the response aswell as the heavy chain isotypes produced. This data allows adetermination of an increase in antibody titer as well as the occurrenceof class switching. Estimates of average affinity are also measured todetermine if affinity maturation occurs during the response to antigen.

After the transgenic bovines have been obtained as described above, theyare utilized to produce transgenic Igs, preferably human, butpotentially that of other species, e.g. dog, cat, non-human primate,other ungulates such as sheep, pig, goat, murines such as mouse, rat,guinea pig, rabbit, etc. As noted, Ig genes are known to be conservedacross species.

Transgenic Antisera and Milk Containing Xenogenous Antibodies

The bovine (or other ungulate) yields transgenic antisera directed towhatever antigen(s) it is endogenously exposed, or to exogenouslyadministered antigen(s). For example, antigens may be administered tothe ungulate to produce desired antibodies reactive with the antigens,including antigens such as pathogens (for example, bacteria, viruses,protozoans, yeast, or fungi), tumor antigens, receptors, enzymes,cytokines, etc. Exemplary pathogens for antibody production include,without limitation, hepatitis virus (for example, hepatitis C),immunodeficiency virus (for example, HIV), herpes virus, parvovirus,enterovirus, ebola virus, rabies virus, measles virus, vaccinia virus,Streptococcus (for example, Streptococcus pneumoniae), Haemaphilus (forexample, Haemophilus influenza), Neisseria (for example, Neisseriameningitis), Coryunebacterium diptheriae, Haemophilus (for example,Haemophilus pertussis), Clostridium (for example, Clostridiumbotulinium), Staphlococcus, Pseudomonas (for example, Pseudomonasaeruginosa), and respiratory syncytial virus (RSV).

One or more pathogens may be administered to a transgenic ungulate togenerate hyperimmune serum useful for the prevention, stabilization, ortreatment of a specific disease. For example, pathogens associated withrespiratory infection in children may be administered to a transgenicungulate to generate antiserum reactive with these pathogens (e.g.,Streptococcus pneumoniae, Haemophilus influenza, and/or Neissariameningitis). These pathogens may optionally be treated to reduce theirtoxicity (e.g., by exposure to heat or chemicals such as formaldehyde)prior to administration to the ungulate.

For the generation of broad spectrum Ig, a variety of pathogens (e.g.,multiple bacterial and/or viral pathogens) may be administered to atransgenic ungulate. This hyperimmune serum may be used to prevent,stabilize, or treat infection in mammals (e.g., humans) and isparticularly useful for treating mammals with genetic or acquiredimmunodeficiencies.

In addition, antibodies produced by the methods of the invention may beused to suppress the immune system, for example, to treat neuropathies,as well as to eliminate particular human cells and modulate specificmolecules. For example, anti-idiotypic antibodies (i.e., antibodieswhich inhibit other antibodies) and antibodies reactive with T cells, Bcells, or cytokines may be useful for the treatment of autoimmunedisease or neuropathy (e.g., neuropathy due to inflammation). Theseantibodies may be obtained from transgenic ungulates that have not beenadministered an antigen, or they may be obtained from transgenicungulates that have been administered an antigen such as a B cell, Tcell, or cytokine (e.g., TNFα).

Transgenic antisera generated from transgenic ungulates that have notbeen administered an antigen may be used to manufacture pharmaceuticalscomprising human polyclonal antibodies, preferably human IgG molecules.These human antibodies may be used in place of antibodies isolated fromhumans as Intraveneous Immunoglobulin (IVIG) therapeutics.

Transgenic antiserum may optionally be enriched for antibodies reactiveagainst one or more antigens of interest. For example, the antiserum maybe purified using standard techniques such as those described by Ausubelet al. (Current Protocols in Molecular Biology, volume 2, p.11.13.1–11.13.3, John Wiley & Sons, 1995). Preferred methods ofpurification include precipitation using antigen or antibody coatedbeads, column chromatography such as affinity chromatography, magneticbead affinity purification, and panning with a plate-bound antigen.Additionally, the transgenic antiserum may be contacted with one or moreantigens of interest, and the antibodies that bind an antigen may beseparated from unbound antibodies based on the increased size of theantibody/antigen complex. Protein A and/or protein G may also be used topurify IgG molecules. If the expression of endogenous antibodies is noteliminated, protein A and/or an antibody against human Ig light chainlambda (Pharmingen) may be used to separate desired human antibodiesfrom endogenous ungulate antibodies or ungulate/human chimericantibodies. Protein A has higher affinity for human Ig heavy chain thanfor bovine Ig heavy chain and may be used to separate desired Igmolecules containing two human heavy chains from other antibodiescontaining one or two ungulate heavy chains. An antibody against humanIg light chain lambda may be used to separate desired Ig moleculeshaving two human Ig lambda chains from those having one or two ungulateIg light chains. Additionally or alternatively, one or more antibodiesthat are specific for ungulate Ig heavy or light chains may be used in anegative selection step to remove Ig molecules containing one or twoungulate heavy and/or light chains.

The resultant antisera may itself be used for passive immunizationagainst an antigen. Alternatively, the antisera has diagnostic,prophylactic, or purification use, e.g. for attaining purification ofantigens.

Alternatively, after antisera administration, B cells may be isolatedfrom the transgenic bovine and used for hybridoma preparation. Forexample, standard techniques may be used to fuse a B cell from atransgenic ungulate with a myeloma to produce a hybridoma secreting amonoclonal antibody of interest (Mocikat, J. Immunol. Methods225:185–189, 1999; Jonak et al., Hum. Antibodies Hybridomas 3:177–185,1992; Srikumaran et al., Science 220:522, 1983). Preferred hybridomasinclude those generated from the fusion of a B-cell with a myeloma froma mammal of the same genus or species as the transgenic ungulate. Otherpreferred myelomas are from a Balb/C mouse or a human. In this instance,hybridomas are provided that make xenogenous monoclonal antibodiesagainst a particular antigen. For example, this technology may be usedto produce human, cat, dog, etc. (dependent upon the specific artificialchromosome) monoclonal antibodies that are specific to pathogens.Methods for selecting hybridomas that produce antibodies havingdesirable properties, i.e., enhanced binding affinity, avidity, are wellknown.

Alternatively, a B cell from a transgenic ungulate may be geneticallymodified to express an oncogene, such as ras, myc, abl, bcl2, or neu, orinfected with a transforming DNA or RNA virus, such as Epstein Barrvirus or SV40 virus (Kumar et al., Immunol. Lett. 65:153–159, 1999;Knight et al., Proc. Nat. Acad. Sci. USA 85:3130–3134, 1988; Shammah etal., J. Immunol. Methods /160-19-25, 1993; Gustafsson and Hinkula, Hum.Antibodies Hybridomas 5:98–104, 1994; Kataoka et al., Differentiation62:201–211, 1997; Chatelut et al., Scand. J. Immunol. 48:659–666, 1998).The resulting immortalized B cells may also be used to produce atheoretically unlimited amount of antibody. Because Ig is also secretedinto the milk of ungulates, ungulate milk may also be used as a sourceof xenogenous antibodies.

While the invention has been described adequately supra, the followingexamples are additionally provided as further exemplification of theinvention.

EXAMPLE 1 Bovine IgM Knock Out

The following procedures were used to generate bovine fibroblast celllines in which one allele of the immunoglobulin heavy chain (mu) locusis disrupted by homologous recombination. A DNA construct for effectingan IgM knockout was generated by the removal of exons 1–4 of the Mulocus (corresponds to IgM heavy chain gene) which were replaced with acopy of a neomycin resistance gene. Using this construct, neomycinresistant cell lines have been obtained which were successfully used innuclear transfer procedures, and blastocysts from these cell lines havebeen implanted into recipient cows. Additionally, some of theseblastocysts were tested to confirm that targeted insertion occurredappropriately in the mu locus using PCR procedures. Blastocystsresulting from nuclear transfer procedures from several of the celllines obtained indicated that heterozygous IgM-KO fetuses were ingestation. Additionally, both male and female cell lines that comprise asingle IgM heavy chain (mu) knockout have been produced. It isanticipated that mating of animals cloned from these cell lines willgive rise to progeny wherein both copies of mu are inactivated. Theseprocedures are discussed in greater detail below.

DNA Construct The DNA used in all transfections described in thisdocument was generated as follows. The four main exons (excluding thetransmembrane domain exons), CH1–4, are flanked by an XhoI restrictionsite at the downstream (CH4) end and an XbaI site at the upstream (CH1)end. The construct used for the transfection procedure consisted of 1.5kb of genomic sequence downstream of the XhoI site and 3.1 Kb of genomicsequence upstream of the XbaI site (FIGS. 3D and 3E). These sequenceswere isolated as described herein from a Holstein cow from a dairy herdin Massachusetts. A neomycin resistance marker was inserted betweenthese two fragments on a 3.5 Kb fragment, replacing 2.4 Kb of DNA,originally containing CH1–4, from the originating genomic sequence. Thebackbone of the vector was pBluescriptIl SK+ (Stratagene) and the insertof 8.1 Kb was purified and used for transfection of bovine fetalfibroblasts. This construct is shown in FIGS. 3A–3C. Other mu knockoutconstructs containing other homologous regions and/or containing anotherantibiotic resistance gene may also be constructed using standardmethods and used to mutate an endogenous mu heavy chain gene.

Transfection /Knockout Procedures Transfection of fetal bovine wasperformed using a commercial reagent, Superfect Transfection Reagent(Qiagen, Valencia, Calif., USA), Catalog Number 301305.

Bovine fibroblasts were generated from disease-tested male Charlaiscattle at Hematech's Kansas facility and sent to Hematech's WorcesterMolecular Biology Labs for use in all experiments described. Any otherungulate breed, genus, or species may be used as the source of donorcells (e.g., somatic cells such as fetal fibroblasts). The donor cellsare genetically modified to contain a mutation that reduces oreliminates the expression of functional, endogenous Ig.

The medium used for culture of bovine fetal fibroblasts consisted of thefollowing components: 500 ml Alpha MEM (Bio-Whittaker # 12-169F); 50 mlfetal calf serum (Hy-Clone #A-1111-D); 2 ml antibiotic/antimyotic(Gibco/BRL #15245-012); 1.4 ml 2-mercaptoethanol (Gibco/BRL #21985-023);5.0 ml L-Glutamine (Sigma Chemical #G-3126); and 0.5 ml tyrosinetartrate (Sigma Chemical #T-6134).

On the day prior to transfection procedures, cells were seeded in 60 mmtissue culture dishes with a targeted confluency of 40–80% as determinedby microscopic examination.

On the day of transfection, 5 μg of DNA, brought to a total volume of150 μl in serum-free, antibiotic-free medium, was mixed with 20 μl ofSuperfect transfection reagent and allowed to sit at room temperaturefor 5–10 minutes for DNA-Superfect complex formation. While the complexformation was taking place, medium was removed from the 60 mm tissueculture dish containing bovine fibroblasts to be transfected, and cellswere rinsed once with 4 ml of phosphate-buffered saline. One milliliterof growth medium was added to the 170 μl DNA/Superfect mixture andimmediately transferred to the cells in the 60 mm dish. Cells wereincubated at 38.5° C., 50% carbon dioxide for 2.5 hours. Afterincubation of cells with the DNA/Superfect complexes, medium wasaspirated off and cells were washed four times with 4 ml PBS. Five ml ofcomplete medium were added and cultures were incubated overnight at38.5° C., 5% CO₂. Cells were then washed once with PBS and incubatedwith one ml of 0.3% trypsin in PBS at 37° C. until cells were detachedfrom the plate, as determined by microscopic observation. Cells fromeach 60 mm dish were split into 24 wells of a 24 well tissue cultureplate (41.7 ul/well). One milliliter of tissue culture medium was addedto each well and plates were allowed to incubate for 24 hours at 38.5°C. and 5% CO₂ for 24 hours.

During all transfection procedures, sham transfections were performedusing a Superfect/PBS mixture containing no DNA, as none of those cellswould be expected to contain the neomycin resistance gene and all cellswould be expected to die after addition of G418 to the tissue culturemedium. This served as a negative control for positive selection ofcells that received DNA.

After the 24 hour incubation, one more milliliter of tissue culturemedium containing 400 μg G418 was added to each well, bringing the finalG418 concentration to 200 μg/ml. Cells were placed back into theincubator for 7 days of G418 selection. During that period, bothtransfected and sham transfection plates were monitored for cell deathand over 7 days, the vast majority of wells from the sham transfectionscontained few to no live cells while plates containing cells thatreceived the DNA showed excellent cell growth.

After the 7 day selection period, the cells from wells at 90–100%confluency were detached using 0.2 ml 0.3% trypsin in PBS and weretransferred to 35 mm tissue culture plates for expansion and incubateduntil they became at least 50% confluent, at which point, cells weretrypsinized with 0.6 ml 0.3% trypsin in PBS. From each 35 mm tissueculture plate, 0.3 ml of the 0.6 ml cell suspension was transferred to a12.5 cm² tissue culture flask for further expansion. The remaining 0.3ml was reseeded in 35 mm dishes and incubated until they attained aminimal confluency of approximately 50%, at which point cells from thoseplates were processed for extraction of DNA for PCR analysis. Flasksfrom each line were retained in the incubator until they had undergonethese analyses and were either terminated if they did not contain thedesired DNA integration or kept for future nuclear transfer andcryopreservation.

Screening for Targeted Integrations As described above the DNA sourcefor screening of transfectants containing the DNA construct was a 35 mmtissue culture dish containing a passage of cells to be analyzed. DNAwas prepared as follows and is adapted from a procedure published byLaird et al. (Laird et al., “Simplified mammalian DNA isolationprocedure”, Nucleic Acids Research, 19:4293). Briefly, DNA was preparedas follows. A cell lysis buffer was prepared with the followingcomponents: 100 mM Tris-HCl buffer, pH 8.5; 5 mM EDTA, pH 8.0; 0.2%sodium dodecyl sulfate; 200 mM NaCl; and 100 ug/ml Proteinase K.

Medium was aspirated from each 35 mm tissue culture dish and replacedwith 0.6 ml of the above buffer. Dishes were placed back into theincubator for three hours, during which time cell lysis and proteindigestion were allowed to occur. Following this incubation, the lysatewas transferred to a 1.5 ml microfuge tube and 0.6 ml of isopropanol wasadded to precipitate the DNA. Tubes were shaken thoroughly by inversionand allowed to sit at room temperature for 3 hours, after which the DNAprecipitates were spun down in a microcentrifuge at 13,000 rpm for tenminutes. The supernatant from each tube was discarded and the pelletswere rinsed with 70% ethanol once. The 70% ethanol was aspirated off andthe DNA pellets were allowed to air-dry. Once dry, each pellet wasresuspended in 30–50 ul of Tris (10 mM)-EDTA (1 mM) buffer, pH 7.4 andallowed to hydrate and solubilize overnight. 5–7 microliters of each DNAsolution was used for each polymerase chain reaction (PCR) procedure.

Two separate PCR procedures were used to analyze transfectants. Thefirst procedure used two primers that were expected to anneal to sitesthat are both located within the DNA used for transfection. The firstprimer sequence is homologous to the neomycin resistance cassette of theDNA construct and the second is located approximately 0.5 Kb away,resulting in a short PCR product of 0.5 Kb. In particular, primers Neol(5′-CTT GAA GAC GAA AGG GCC TCG TGA TAC GCC-3′, SEQ ID NO: 42) andIN2521 (5′-CTG AGA CTT CCT TTC ACC CTC CAG GCA CCG-3′, SEQ ID NO: 43)were used. A Qiagen PCR kit was used for this PCR reaction. The PCRreaction mixture contained 1 pmole of each primer, 5 ul of 10× reactionbuffer, 10 ul of Q solution, 5 ul of DNA, and 1 ul of dNTP solution. Thereaction mixture was brought to a total volume of 50 ul with H₂O. ThisPCR amplification was performed using an initial denaturing incubationat 94° C. for two minutes. Then, 30 cycles of denaturation, annealing,and amplification were performed by incubation at 94° C. for 45 seconds,60° C. for 45 seconds, and 72° C. for two minutes. Then, the reactionmixture was incubated at 72° C. for five minutes and at 4° C. until themixture was removed from the PCR machine. Alternatively, any otherprimers that are homologous to the region of the knockout construct thatintegrates into the genome of the cells may be used in a standard PCRreaction under appropriate reaction conditions to verify that cellssurviving G418 selection were resistant as a result of integration ofthe DNA construct.

Because only a small percentage of transfectants would be expected tocontain a DNA integration in the desired location (the Mu locus),another pair of primers was used to determine not only that the DNAintroduced was present in the genome of the transfectants but also thatit was integrated in the desired location. The PCR procedure used todetect appropriate integration was performed using one primer locatedwithin the neomycin resistance cassette of the DNA construct and oneprimer that would be expected to anneal over 1.8 Kb away, but only ifthe DNA had integrated at the appropriate site of the IgM locus (sincethe homologous region was outside the region included in the DNAconstruct used for transfection). The primer was designed to anneal tothe DNA sequence immediately adjacent to those sequences represented inthe DNA construct if it were to integrate in the desired location (DNAsequence of the locus, both within the region present in the DNAconstruct and adjacent to them in the genome was previously determined).In particular, primers Neol and OUT3570 (5′-CGA TGA ATG CCC CAT TTC ACCCAA GTC TGT C-3′, SEQ ID NO: 44) were used for this analysis. This PCRreaction was performed using a Qiagen PCR kit as described above for thefirst PCR reaction to confirm the integration of the targeting constructinto the cells. Alternatively, this PCR analysis may be performed usingany appropriate reaction conditions with any other primer that ishomologous to a region of the knockout construct that integrates intothe genome of the cells and any other primer that is homologous to aregion in the genome of the cells that is upstream or downstream of thesite of integration.

Using these methods, 135 independent 35 mm plates were screened fortargeted integration of the DNA construct into the appropriate locus. Ofthose, DNA from eight plates was determined to contain an appropriatelytargeted DNA construct and of those, three were selected for use innuclear transfer procedures. Those cells lines were designated as“8-1C”, “5-3C” and “10-1C”. Leftover blastocysts not used for transferinto recipient cows were used to extract DNA which was subjected toadditional PCR analysis. This analysis was effective using a nested PCRprocedure using primers that were also used for initial screening oftransfected lines.

As noted above, three cell lines were generated using the gene targetingconstruct designed to remove exons 1–4 of the mu locus. These lines alltested positive for targeted insertions using a PCR based test and wereused for nuclear transfers. Leftover blastocysts resulting from thosenuclear transfers were screened by PCR testing the appropriatelytargeted construct. The following frequencies of positive blastocystswere obtained:

Cell Line 8-1C: 6/8 Cell Line 10-1C: 2/16 Cell Line 5-3C: 0/16

Although at forty days of gestation, 11 total pregnancies were detectedby ultrasound, by day 60, 7 fetuses had died. The remaining 4 fetuseswere processed to regenerate new fetal fibroblasts and remaining organswere used to produce small tissue samples for PCR analysis. The resultsof the analyses are below:

Line 8-1C: two fetuses, one fetus positive for targeted insertion by PCRLine 10-1C: one fetus, positive for targeted insertion by PCR Line 5-3C:one fetus, negative for targeted insertion by PCR

Surprisingly, although the frequency of 10-1 C blastocysts testingpositive for targeted insertion was only 2/16, the one viable 60-dayfetus obtained from that cell line was positive as determined by PCR. Apositive fetus from 8-1C was also obtained. Southern blot analysis ofDNA of all tissue samples is being effected to verify that the constructnot only targeted correctly at one end (which is determined by PCR ofthe shorter region of homology present in the original construct) butalso at the other end. Based on results to date, it is believed that twoheavy chain knockout fetuses from two independent integration eventshave been produced. Also, since these fetuses were derived from twodifferent lines, at least one is likely to have integrated constructcorrectly at both ends. Once the Southern blot analyses have confirmedappropriate targeting of both ends of targeting construct, furthernuclear transfers will be performed to generate additional fetuses whichwill be carried to term.

Nuclear Transfer and Embryo Transfer Nuclear transfers were performedwith the K/O cell line (8-1-C (18)) and eight embryos were produced. Atotal of six embryos from this batch were transferred to three diseasefree recipients at Trans Ova Genetics (“TOG”; Iowa).

Frozen embryos have been transferred to ten disease free recipients toobtain disease free female fibroblast cell lines. Fetal recoveries arescheduled after confirming the pregnancies at 35–40 days.

Pregnancy Diagnosis and Fetal Recovery Pregnancy status of the eighteenrecipients transferred with cloned embryos from knockout fetal cells waschecked by ultrasonography. The results are summarized below.

TABLE 2 Pregnancy at 40 days using mu heavy chain knockout donor cellsClone ID No of recips transferred Pregnancy at 40 days (%) 8-1-0C 5 4(80) 10-1-C 6 4 (67) 5-3-C 5 3 (60) Total 16 11 (69) 

Pregnancy Diagnosis Pregnancy status of the three recipients to whomcloned embryos were transferred from knockout cells (8-1C) was checked;one was open and the other two required reconfirmation after one month.

Fetal Recoveries and Establishment of Cell Lines Eleven pregnancies withthe K/O embryos at 40 days were obtained. Four live fetuses were removedout of these at 60 days. Cell lines were established from all four andcryopreserved for future use. Also we collected and snap froze tissuesamples from the fetuses and sent them to Hematech molecular biologylaboratory for PCR/Southern blot analysis.

All four of the cell lines were male. In order to secure a female cellline, cell lines were established and cryopreserved for futureestablishment of K/O cells from the fetuses (six) collected at 55 daysof gestation from the pregnancies established at Trans Ova Genetics withdisease free recipients. Recently, the existence of a female cell linecontaining a mu knockout was confirmed. This female cell line may beused to produce cloned animals which may be mated with animals generatedfrom the male cell lines, and progeny screened for those that containthe double mu knockout.

EXAMPLE 2 Introduction of HAC

Additional experiments were carried out to demonstrate thatimmunoglobulin heavy chain (mu) and lambda light chain may be producedby a bovine host, either alone or in combination. In addition, theseexperiments demonstrated that the immunoglobulin chains were rearrangedand that polyclonal sera was obtained. In these procedures,immunoglobulin-expressing genes were introduced into bovine fibroblastsusing human artificial chromosomes. The fibroblasts were then utilizedfor nuclear transfer, and fetuses were obtained and analyzed forantibody production. These procedures and results are described in moredetail below.

HAC Constructs The human artificial chromosomes (HACs) were constructedusing a previously described chromosome-cloning system (Kuroiwa et al.,Nature Biotech. 18: 1086–1090, 2000). Briefly, for the construction ofΔHAC, the previously reported human chromosome 22 fragment (hChr22)containing a loxP sequence integrated at the HCF2 locus was truncated atthe AP000344 locus by telomere-directed chromosomal truncation (Kuroiwaet al., Nucleic Acid Res., 26: 3447–3448, 1998). Next, cell hybrids wereformed by fusing the DT40 cell clone containing the above hChr22fragment (hCF22) truncated at the AP000344 locus with a DT40 cell clone(denoted “R clone”) containing the stable and germline-transmittablehuman minichromosome SC20 vector. The SC20 vector was generated byinserting a loxP sequence at the RNR2 locus of the S20 fragment. TheSC20 fragment is a naturally-occurring fragment derived from humanchromosome 14 that includes the entire region of the human Ig heavychain gene (Tomizuka et al., Proc. Natl. Acad. Sci. USA 97:722, 2000).The resulting DT40 cell hybrids contained both hChr fragments. The DT40hybrids were transfected with a Cre recombinase-expression vector toinduce Cre/loxP-mediated chromosomal translocation between hCF22 and theSC20 vector. The stable transfectants were analyzed using nested PCR toconfirm the cloning of the 2.5 megabase hChr22 region, defined by theHCF2 and AP000344 loci, into the loxP-cloning site in the SC20 vector.The PCR-positive cells which were expected to contain ΔHAC were thenisolated by FACS sorting based on the fluorescence of the encoded greenfluorescent protein. Fluorescent in situ hybridization (FISH) analysisof the sorted cells was also used to confirm the presence of ΔHAC, whichcontains the 2.5 megabase hChr22 insert.

Similarly, ΔΔHAC was also constructed using this chromosome-cloningsystem. The hChr22 fragment was truncated at the AP000344 locus, andthen the loxP sequence was integrated into the AP000553 locus byhomologous recombination in DT40 cells. The resulting cells were thenfused with the R clone containing the SC20 minichromosome vector. Thecell hybrids were transfection with a Cre-expression vector to allowCre/loxP-mediated chromosomal translocation. The generation of ΔΔHAC,which contains the 1.5 megabase hChr22 insert, defined by the AP000553and AP000344 loci, was confirmed by PCR and FISH analyses.

The functionality of ΔHAC and ΔΔHAC in vivo was assessed by thegeneration of chimeric mice containing these HACs. These HACs wereindividually introduced into mouse embryonic stem (ES) cells, which werethen used for the generation of chimeric mice using standard procedures(Japanese patent number 2001–142371; filed May 11, 2000). The resultingmice had a high degree of chimerism (85–100% of coat color),demonstrating a high level of pluripotency of the ES cells containingthese HACs and the mitotic stability of these HACs in vivo. Furthermore,ΔHAC was transmitted through the germline of the ΔHAC chimeric mouse tothe next offspring, demonstrating the meiotic stability of this HAC.

Chicken DT40 cells retaining these HACs have been deposited under theBudapest treaty on May 9, 2001 in the International Patent OrganismDepository, National Institute of Advanced Industrial Science andTechnology, Tsukuba Central 6, 1-1, Higashi 1-Chome Tsukuba-shi,Ibaraki-ken, 305–8566 Japan. The depository numbers are as follows: ΔHAC(FERM BP-7582), ΔΔHAC (FERM BP-7581), and SC20 fragment (FERM BP-7583).Chicken DT40 cells retaining these HACs have also been deposited in theFood Industry Research and Development Institute (FIRDI) in Taiwan. Thedepository numbers and dates are as follows: ΔHAC (CCRC 960144; Nov. 9,2001), ΔΔHAC (CCRC 960145; Nov. 9, 2001), and SC20 fragment (the cellline was deposited under the name SC20 (D); CCRC 960099; Aug. 18, 1999).

The 2.5 megabase (Mb) hChr22 insert in ΔHAC is composed of the followingBAC contigs, which are listed by Genbank accession number: AC002470,AC002472, AP000550, AP000551, AP000552, AP000556, AP000557, AP000558,AP000553, AP000554, AP000555, D86995, D87019, D87012, D88268, D86993,D87004, D87022, D88271, D88269, D87000, D86996, D86989, D88270, D87003,D87018, D87016, D86999, D87010, D87009, D87011, D87013, D87014, D86991,D87002, D87006, D86994, D87007, D87015, D86998, D87021, D87024, D87020,D87023, D87017, AP000360, AP00361, AP000362, AC000029, AC000102, U07000,AP000343, and AP000344. The 1.5 Mb hChr22 insert in ΔΔHAC is composed ofthe following BAC contigs: AP000553, AP000554, AP000555, D86995, D87019,D87012, D88268, D86993, D87004, D87022, D88271, D88269, D87000, D86996,D86989, D88270, D87003, D87018, D87016, D86999, D87010, D87009, D87011,D87013, D87014, D86991, D87002, D87006, D86994, D87007, D87015, D86998,D87021, D87024, D87020, D87023, D87017, AP000360, AP00361, AP000362,AC000029, AC000102, U07000, AP000343, and AP000344 (Dunham et al, Nature402:489–499, 1999).

Generation of Bovine Fetal Fibroblasts To generate bovine fetalfibroblasts, day 45 to 60 fetuses were collected from disease-testedHolstein or Jersey cows housed at Trans Ova (Iowa), in which thepedigree of the male and female parents were documented for threeconsecutive generations. The collected fetuses were shipped on wet iceto Hematech's Worcester Molecular Biology Division for the generation ofprimary fetal fibroblasts. Following arrival, the fetus(es) weretransferred to a non-tissue culture grade, 100 mm plastic petri dish ina tissue culture hood. Using sterile forceps and scissors, theextraembryonic membrane and umbilical cord were removed from the fetus.After transferring the fetus to a new plastic petri dish, the head,limbs and internal organs were removed. The eviscerated fetus wastransferred to a third petri dish containing approximately 10 ml offetus rinse solution composed of: 125 ml 1× Dulbecco's-PBS (D-PBS) withCa²⁺ and Mg²⁺ (Gibco-BRL, cat#. 14040); 0.5 ml Tylosine Tartrate (8mg/ml, Sigma, cat#. T-3397); 2 ml Penicillin-Streptomycin (Sigma, cat#.P-3539); and 1 ml of Fungizone (Gibco-BRL, cat#. 15295-017) (mixed andfiltered through a 0.2 μm nylon filter unit [Nalgene, cat#. 150-0020).

The fetus was washed an additional three times with the fetus rinsesolution to remove traces of blood, transferred to a 50 ml conicaltissue culture tube, and finely minced into small pieces with a sterilescalpel. The tissue pieces were washed once with 1×D-PBS without Ca²⁺and Mg²⁺ (Gibco-BRL, cat#. 14190). After the tissue pieces settled tothe bottom of the tube, the supernatant was removed and replaced withapproximately 30 ml of cell dissociation buffer (Gibco-BRL, cat#.13151-014). The tube was inverted several times to allow mixing andincubated at 38.5° C./5% CO₂ for 20 minutes in a tissue cultureincubator. Following settling of the tissue to the bottom of the tube,the supernatant is removed and replaced with an equivalent volume offresh cell dissociation buffer. The tissue and cell dissociation buffermixture was transferred to a sterile, 75 ml glass trypsinizing flask(Wheaton Science Products, cat#. 355393) containing a 24 mm,round-ended, spin bar. The flask was transferred to a 38.5° C./5% CO₂tissue culture incubator, positioned on a magnetic stir plate, andstirred at a sufficient speed to allow efficient mixing forapproximately 20 minutes. The flask was transferred to a tissue culturehood; the tissue pieces allowed to settle, followed by removal of thesupernatant and harvesting of the dissociated cells by centrifugation at1,200 rpm for five minutes. The cell pellet was re-suspended in a smallvolume of complete fibroblast culture media composed of: 440 ml alphaMEM (BioWhittaker, cat#. 12-169F); 50 ml irradiated fetal bovine serum;5 ml GLUTAMAX-I supplement (Gibco-BRL, cat#. 25050-061); 5 mlPenicillin-Streptomycin (Sigma, cat#. P-3539); 1.4 ml 2-mercaptoethanol(Gibco-BRL, cat#. 21985-023) (all components except the fetal bovineserum were mixed were filtered through 0.2 μm nylon filter unit[Nalgene, cat#. 151-4020]), and stored on ice. The dissociation processwas repeated three additional times with an additional 30 ml of celldissociation solution during each step. Cells were pooled; washed incomplete fibroblast media; passed sequentially through 23 and 26 gaugeneedles, and finally through a 70 μm cell strainer (B-D Falcon, cat#.352350) to generate a single cell suspension. Cell density and viabilitywere determined by counting in a hemacytometer in the presence of trypanblue (0.4% solution, Sigma, cat#. T-8154).

Primary fibroblasts were expanded at 38.5° C./5% CO₂ in completefibroblast media at a cell density of 1×10⁶ viable cells per T75 cm²tissue culture flask. After 3 days of culture or before the cellsreached confluency, the fibroblasts were harvested by rinsing the flaskonce with 1×D-PBS (without Ca²⁺ and Mg²⁺) and incubating with 10 ml ofcell dissociation buffer for 5 to 10 minutes at room temperature.Detachment of cells was visually monitored using an inverted microscope.At this step, care was taken to ensure that cell clumps weredisaggregated by pipeting up-and-down. After washing and quantitation,the dissociated fibroblasts were ready for use in gene targetingexperiments. These cells could also be cryopreserved for long-termstorage.

Introduction of HACs into Bovine Fetal Fibroblasts ΔHAC and ΔΔHAC weretransferred from the DT40 cell hybrids to Chinese hamster ovary (CHO)cells using microcell-mediated chromosome transfer (MMCT) (Kuroiwa etal. Nature Biotech. 18: 1086–1090, 2000). The CHO clone containing ΔHAC(“D15 clone”) was cultured in F12 (Gibco) medium supplemented with 10%FBS (Gibco), 1 mg/ml of G418, and 0.2 mg/ml of hygromycin B at 37° C.and 5% CO₂. The D15 clone was expanded into twelve T25 flasks. When theconfluency reached 80–90%, colcemid (Sigma) was added to the medium at afinal concentration of 0.1 μg/ml. After three days, the medium wasexchanged with DMEM (Gibco) supplemented with 10 μg/ml of cytochalacin B(Sigma). The flasks were centrifuged for 60 minutes at 8,000 rpm tocollect microcells. The microcells were purified through 8, 5, and 3-μmfilters (Costar) and then resuspended in DMEM medium. The microcellswere used for fusion with bovine fibroblasts as described below.

Bovine fetal fibroblasts were cultured in α-MEM (Gibco) mediumsupplemented with 10% FBS (Gibco) at 37° C. and 5% CO₂. The fibroblastswere expanded in a T175 flask. When the confluency reached 70–80%, thecells were detached from the flask with 0.05% trypsin. The fibroblastcells were washed twice with DMEM medium and then overlayed on themicrocell suspension. After the microcell-fibroblast suspension wascentrifuged for five minutes at 1,500 rpm, PEG1500 (Roche) was added tothe pellet according to the manufacturer's protocol to enable fusion ofthe microcells with the bovine fibroblasts. After fusion, the fusedcells were plated into six 24-well plates and cultured in α-MEM mediumsupplemented with 10% FBS for 24 hours. The medium was then exchangedwith medium containing 0.7 mg/ml of G418. After growth in the presenceof the G418 antibiotic for about two weeks, the G418 resistant, fusedcells were selected. These G418-resistant clones were used for nucleartransfer, as described below.

Similarly, ΔΔHAC from the CHO clone ΔΔC13 was transferred into bovinefetal fibroblasts by means of MMCT. The selected G418-resistant cloneswere used for nuclear transfer.

Nuclear Transfer, Activation, and Embryo Culture The nuclear transferprocedure was carried out essentially as described earlier (Cibelli etal., Science 1998: 280:1256–1258). In vitro matured oocytes wereenucleated about 18–20 hours post maturation (hpm) and chromosomeremoval was confirmed by bisBenzimide (Hoechst 33342, Sigma) labelingunder UV light. These cytoplast-donor cell couplets were fused, by usingsingle electrical pulse of 2.4 kV/cm for 20 μsec (Electrocellmanipulator 200, Genetronics, San Diego, Calif.). After 3–4 hrs, arandom sub-set of 25% of the total transferred couplets was removed, andthe fusion was confirmed by bisBenzimide labeling of the transferrednucleus. At 30 hpm reconstructed oocytes and controls were activatedwith calcium ionophore (5 μM) for 4 minutes (Cal Biochem, San Diego,Calif.) and 10 μg Cycloheximide and 2.5 μg Cytochalasin D (Sigma) in ACMculture medium for 6 hours as described earlier (Lin et al., Mol.Reprod. Dev. 1998: 49:298–307; Presicce et al., Mol. Reprod. Dev.1994:38:380–385). After activation eggs were washed in HEPES bufferedhamster embryo culture medium (HECM-Hepes) five times and placed inculture in 4-well tissue culture plates containing irradiated mousefetal fibroblasts and 0.5 ml of embryo culture medium covered with 0.2ml of embryo tested mineral oil (Sigma). Twenty five to 50 embryos wereplaced in each well and incubated at 38.5° C. in a 5% CO₂ in airatmosphere. On day four 10% FCS was added to the culture medium.

Embryo Transfer Day 7 and 8 nuclear transfer blastocysts weretransferred into day 6 and 7 synchronized recipient heifers,respectively. Recipient animals were synchronized using a singleinjection of Lutalyse (Pharmacia & Upjohn, Kalamazoo, Mich.) followed byestrus detection. The recipients were examined on day 30 and day 60after embryo transfer by ultrasonography for the presence of conceptusand thereafter every 30 days by rectal palpation until 270 days. Theretention of a HAC in these bovine fetuses is summarized in Table 3 andis described in greater detail in the sections below.

TABLE 3 Summary of HAC retention in bovine fetuses HAC Recip/ Reten-Cell Fetus Recovery tion HAC Clone No. NT Date Date Fetal Age H L ΔΔ4-12 5580 2/14 4/13  58 + + ΔΔ 2-14 5848 2/15 4/13  57 − − ΔΔ 4-12 ^( 5868A) 2/14 6/13 119 + + ΔΔ 4-12  ^( 5868B) 2/14 6/13 119 + + ΔΔ4-12  ^( 5542A) 2/14 5/16  91 + + ΔΔ 4-12  ^( 5542B) 2/14 5/16  91 + +ΔΔ 4-12 5174 2/14 5/16  91 (abnormal) nd nd ΔΔ 4-12 6097 2/14 Remains160 (7/24) nd nd Δ 4-8  6032 1/31 3/30  58 + + Δ 2-13 5983 2/2  3/30  56− − Δ 4-2  5968 2/2  3/30  56 + + Δ 2-22 6045 2/2  3/30  56 + + Δ 4-8 5846 1/31 4/20  79 − − Δ 2-13 6053 2/2  4/27  84 + − Δ 4-2  5996 2/1 4/20  77 + −

Introduction of a HAC Containing a Fragment of Human Chromosome #14 TheSC20 fragment, a human chromosome #14 fragment (“hchr.14fg”, containingthe Ig heavy chain gene), was introduced into fetal fibroblast cells insubstantially the same manner as described above. Any other standardchromosome transfer method may also be used to insert this HAC oranother HAC containing a human Ig gene into donor cells. The resultingdonor cells may be used in standard nuclear transfer techniques, such asthose described above, to generate transgenic ungulates with the HAC.

The pregnancy status of the 28 recipients to whom cloned embryos weretransferred from cells containing the hchr. 14fg was checked byultrasonography. The results are summarized in Table 4.

TABLE 4 Pregnancy at 40 days using donor cells containing hchr.14fg Noof recips Pregnancy at 40 days Clone ID transferred (%) 2-1 08 03 (38)4-2 10 00 (00) 4-1 05 00 (00) 4-1 03 01 (33) 2-1 02 01 (50) Total 28 05(18)

The pregnancy rates were lower than anticipated. This is believed to beattributable to extremely abnormally hot weather during embryo transfer.

As illustrated in FIG. 27, pregnancy rates for HAC carrying embryosappear to be equivalent to non-transgenic cloned pregnancies. Onerecipient carrying a ΔΔHAC calf gave birth recently to a live healthycalf. Others will be born over the next several months

Demonstration of Rearrangement and Expression of Human Heavy Chain Locusin a ΔHAC Bovine Fetus

Cloned ΔHAC-transgenic bovine fetuses were removed at variousgestational days and analyzed for the presence, rearrangement, andexpression of the human immunoglobulin loci. Analysis of genomic DNA andcDNA obtained by RT-PCR from spleen and nonlymphoid tissues (liver andbrain) of one of these fetuses indicated the presence, rearrangement,and expression of the ΔHAC.

Presence of Human Heavy and/or Light Chain in ΔHAC Fetuses To determinewhether the human heavy and light chains were retained in ΔHAC fetuses,liver DNA was isolated from ΔHAC fetuses and analyzed by PCR for thepresence of genomic DNA encoding human heavy and light chains.

For the detection of genomic heavy chain DNA, the following primers wereused: VH3-F 5′-AGTGAGATAAGCAGTGGATG-3′ (SEQ ID NO: 1) and VH3-R5′-CTTGTGCTACTCCCATCACT-3′ (SEQ ID NO: 2). The primers used fordetection of lambda light chain DNA were IgL-F5′-GGAGACCACCAAACCCTCCAAA-3′ (SEQ ID NO: 3) and IgL-R5′-GAGAGTTGCAGAAGGGGTYGACT-3′ (SEQ ID NO: 4). The PCR reaction mixturescontained 18.9 μl water, 3 μl of 10×Ex Taq buffer, 4.8 μl of dNTPmixture, 10 pmol forward primer and 10 pmol of reverse primer, 1 μl ofgenomic DNA, and 0.3 μl of Ex Taq. Thirty-eight cycles of PCR wereperformed by incubating the reaction mixtures at the followingconditions: 85° C. for three minutes, 94° C. for one minute, 98° C. for10 seconds, 56° C. for 30 seconds, and 72° C. for 30 seconds.

As shown in FIG. 5, fetuses #5968, 6032 and 6045 each contained bothhuman heavy chain (μ) and light chain (λ) loci. Fetus #5996 containedonly the human heavy chain locus. Fetus #5983 did not contain the humanheavy chain and may not have contained the human light chain. Fetus#5846 did not contain either human sequence. Thus, fetuses #5983 and5846 may not have retained the HAC. These results suggested that ΔHACcan be stably retained up to gestational day 58 in bovines.

Presence of Human Cmu Exons in ΔHAC Fetus #5996 Primers specific for amRNA transcript including portions of Cmu 3 and Cmu 4 were used todetermine whether ΔHAC was present and expressing transcripts encodingthe constant region of the human mu locus of fetus #5996.

For this RT-PCR analysis of the genomic constant region of the human muheavy chain, primers “CH3-F1” (5′accacctatgacagcgtgac-3′, SEQ ID NO: 5)and “CH4-R2” (5′-gtggcagcaagtagacatcg-3′, SEQ ID NO: 6) were used togenerate a RT-PCR product of 350 base pairs. This PCR amplification wasperformed by an initial denaturing incubation at 95° C. for fiveminutes. Then, 35 cycles of denaturation, annealing, and amplificationwere performed by incubation at 95° C. for one minute, 59° C. for oneminute, and 72° C. for two minutes. Then, the reaction mixtures wereincubated at 72° C. for 10 minutes. Rearranged bovine heavy chain wasdetected using primers I7L and P9, as described below (FIG. 7). As aninternal control, levels of GAPDH RNA was detected using primers “GAPDHforward” (5′-gtcatcatctctgccccttctg-3′, SEQ ID NO: 7) and “GAPDHreverse” (5′-aacaacttcttgatgtcatcat-3′, SEQ ID NO: 8). For thisamplification of GAPDH RNA, samples were incubated at 95° C. for fiveminutes, followed by 35 cycles of incubation at 95° C. for one minute,55° C. for one minute, and 72° C. for two minutes. Then, the mixtureswere incubated at 72° C. for seven minutes.

This analysis showed that RT-PCR analysis of the spleen of fetus #5996produced a band (lane 3) matching the amplification products generatedusing control human spleen cDNA (lane 4) and cDNA obtained from a ΔHACchimeric mouse (lane 5) (FIG. 6). No such band was detected innonlymphoid tissues: bovine liver (lane 1) or bovine brain (lane 2). Thecapacity of these tissues to support RT-PCR was shown by the successfulamplification of the housekeeping gene, GADPH, in both liver (lane 10 ofFIG. 6) and brain (lane 6 of FIG. 7).

Rearrangement of Bovine Heavy Chain Locus by 77 Gestational Days TheΔHAC fetus #5996 was tested to determine whether it had undergone thedevelopmental processes necessary for the expression and activation ofthe recombination system required for immunoglobulin heavy chain locusrearrangement. For this analysis, standard RT-PCR analysis was performedto detect the presence of mRNA transcripts encoding mu-VHrearrangements. RNA isolated from the spleen, liver, and brain of fetus#5996 was analyzed by RT-PCR using primers “17L”(5′-ccctcctctttgtgctgtca-3′, SEQ ID NO: 9) and “P9”(5′-caccgtgctctcatcggatg-3′, SEQ ID NO: 10). The PCR reaction mixtureswere incubated at 95° C. for 3 minutes, and then 35 cycles ofdenaturation, annealing, and amplification were performed using thefollowing conditions: 95° C. for one minute, 58° C. for one minute, and72° C. for two minutes. The reaction mixture was then incubated at 72°C. for 10 minutes.

Lane 5 of FIG. 7 shows that a product of the size expected foramplification of a rearranged bovine heavy chain (450 base pairs) wasobtained. This product migrated to a position equivalent to that of acontrol bovine Cmu heavy chain cDNA known to contain sequencescorresponding to rearranged bovine heavy chain transcripts (lane 7). Asexpected, the rearranged heavy chain was expressed in the spleen (lane5), but absent from the brain (lane 2) and liver (lane 3) at this pointin development.

Rearrangement and Expression of the Human Heavy Chain Locus in the ΔHACFetus #5996 The rearrangement and expression of the human heavy chainlocus was demonstrated by the amplification of a segment of DNAincluding portions of Cmu and VH regions. Primers specific for RNAtranscripts including portions of Cmu (Cmu1) and VH (VH3-30) were usedto determine if RNA transcripts containing rearranged human Cmu-VDJsequences were present (FIG. 8).

For this RT-PCR analysis, primers “Cmu1” (5′-caggtgcagctggtggagtctgg-3′,SEQ ID NO: 11) and “VH3-30” (5′caggagaaagtgatggagtc-3′, SEQ ID NO: 12)were used to produce a RT-PCR product of 450 base pairs. This RT-PCR wasperformed by incubating reaction mixtures at 95° for 3 minutes, followedby 40 cycles of incubation at 95° for 30 minutes, 69° for 30 minutes,and 72° for 45 minutes, and one cycle of incubation at 72° for 10minutes. This RT-PCR product was then reamplified with the same primersby one cycle of incubation at 95° C. for three minute, 40 cycles ofincubation at 95° C. for one minute, 59° C. for one minute, 72° C. forone minute, and one cycle of incubation at 72° C. for 10 minutes. As aninternal control, RT-PCR amplification of GAPDH was performed asdescribed above.

The gel in FIG. 8 shows that RT-PCR analysis of the spleen from fetus#5996 produced a band (lane 5) matching the amplification productsgenerated using human spleen cDNA (lane 4) or ΔHAC chimeric mouse spleencDNA (lane 1). No such band was detected in bovine liver (lane 2) orbovine brain (lane 3). As a positive control, amplification of GADPH RNA(lanes 8 and 9) showed the capacity of these tissues to support RT-PCR.

Rearrangement and expression of the human heavy chain region in fetus#5996 was also demonstrated by RT-PCR analysis using primers CH3-F3(5′-GGAGACCACCAAACCCTCCAAA-3′, SEQ ID NO: 13) and CH4-R2(5′-GTGGCAGCAAGTAGACATCG-3′, SEQ ID NO: 14). These PCR reaction mixturescontained 18.9 μl water, 3 μl of 10×Ex Taq buffer, 4.8 μl of dNTPmixture, 10 pmol forward primer, 10 pmol of reverse primer, 1 μl ofcDNA, and 0.3 μl of Ex Taq. Forty PCR cycles were performed byincubating the reaction mixtures under the following conditions: 85° C.for three minutes, 94° C. for one minute, 98° C. for 10 seconds, 60° C.for 30 seconds, and 72° C. for 30 seconds.

As shown in lanes 6 and 7 of FIG. 9, an amplified sequence from thespleen of fetus #5996 was the same size as the spliced constant regionfragments from the two positive controls: a sample from a human spleen(lane 8) and a ΔHAC chimeric mouse spleen (lane 9). As expected, thenegative controls from a normal mouse spleen and a bovine spleen did notcontain an amplified sequence (lanes 1 and 2). Samples from the liverand brain of fetus #5996 did not contain an amplified spliced sequenceof the same size as the spliced human mu heavy chain constant regionfragments but did contain a amplified sequence of an unspliced genomicfragment derived from genomic DNA contaminating the RNA sample (lanes 3,4, and 5).

VDJ Rearrangement of the Human Heavy Chain Locus in a ΔHAC Fetus RT-PCRanalysis was also performed to further demonstrate VDJ rearrangement inthe heavy chain locus in ΔHAC fetus #5996. Nested RT-PCR was performedusing primer Cmu-1 (5′-CAGGAGAAAGTGATGGAGTC-3′, SEQ ID NO: 15) for thefirst reaction, primer Cmu-2 (5′-AGGCAGCCAACGGCCACGCT-3′, SEQ ID NO: 16)for the second reaction, and primer VH3-30.3(5′-CAGGTGCAGCTGGTGGAGTCTGG-3′, SEQ ID NO: 17) for both reactions. TheRT-PCR reaction mixtures contained 18.9 μl water, 3 μl of 10×Ex Taqbuffer, 4.8 μl of dNTP mixture, 10 pmol forward primer, 10 pmol ofreverse primer, 1 μl of cDNA, and 0.3 μl of Ex Taq. The RT-PCR wasperformed using 38 cycles under the following conditions for the firstreaction: 85° C. for three minutes, 94° C. for one minute, 98° C. for 10seconds, 65° C. for 30 seconds, and 72° C. for 30 seconds. For thesecond reaction, 38 cycles were performed under the followingconditions: 85° C. for three minutes, 94° C. for one minute, 98° C. for10 seconds, 65° C. for 30 seconds, and 72° C. for 30 seconds usingprimers VH3-30.3 and Cmu-2 (5′-AGGCAGCCAACGGCCACGCT-3′, SEQ ID NO: 16).

As shown in lanes 6 and 7 of FIG. 10, RT-PCR analysis of the spleen offetus #5996 produced a heavy chain band of the same size as the positivecontrols in lanes 8 and 9. Samples from the liver and brain of fetus#5996 contained some contaminating rearranged DNA (lanes 3 and 5). Thenegative controls in lanes 1 and 2 produced bands of the incorrect size.

Verification of ΔHAC Rearrangement By Sequencing The cDNA obtained byreverse transcription of RNA from the spleen of the ΔHAC fetus #5996 wasamplified with primers specific for rearranged human mu and run on anagarose gel. The band produced by amplification with the Cmu1-VH3-30primer pair was excised from the gel. The amplified cDNA was recoveredfrom the band and cloned. DNA from a resulting clone that wasPCR-positive for rearranged human mu was purified and sequenced (FIG.11A).

The sequence from this ΔHAC fetus is greater than 95% homologous to over20 known human heavy chain sequences. For example, the mu chain of ahuman anti-pneumococcal antibody is 97% homologous to a region of thissequence (FIG. 11B).

Additional sequences from rearranged human heavy chains were alsoobtained by RT-PCR analysis of the spleen of fetus #5996 using primersCmu-1 and VH3-30.3, followed by reamplification using primers Cmu-2 andVH3-30.3. The RT-PCR products were purified using CHROMA SPIN column(CLONETECH) and cloned into the pCR2.1 TA-cloning vector (Invitrogen)according to manufacturer's protocol. The Dye Terminator sequencereaction (ABI Applied System) was performed in a 10 μl volume reactionmixture composed of BigDye Terminator reaction mixture (3 μl), templateplasmid (200 ng), and the Cmu-2 primer (1.6 pmol). The sequencingreaction was performed using a ABI 3700 sequencer. For this analysis,twenty-five cycles were conducted under the following conditions: 96° C.for one minute, 96° C. for 10 seconds, 55° C. for five seconds, and 60°C. for four minutes.

At least two rearranged human heavy chain transcripts were identified,which were VH3-11/D7-27/JH3/Cμ and VH3-33/D6-19/JH2/Cμ (FIGS. 12A and12B). The results demonstrate that VDJ rearrangement of the human muheavy chain locus occurs in the ΔHAC in the spleen of fetus #5996. Theidentification of more than one rearranged heavy chain sequence from thesame fetus also demonstrates the ability of ΔHAC fetuses to generatediverse human immunoglobulin sequences.

Rearrangement and Expression of Human Heavy and Light Chain Loci inΔΔHAC Fetus

Cloned fetuses derived from bovine fetal fibroblasts transchromosomalfor the ΔΔHAC were removed from recipient cows at various gestationaldays. The fetuses were analyzed for the presence and rearrangement ofthe HAC-borne human immunoglobulin heavy and lambda light chain loci.Studies of genomic DNA from these tissues indicated the presence of thehuman immunoglobulin heavy and light chains in some of the fetuses.Examination of cDNA derived from the spleens of these fetuses indicatedrearrangement and expression of the immunoglobulin heavy and light chainloci in some of these fetuses. FACS analysis also demonstrated theexpression of human lambda light chain protein on the surface of spleniclymphocytes in two of the fetuses.

Presence of Human Heavy and Light Chain Loci in ΔΔHAC Fetuses Todetermine whether ΔΔHAC fetuses retained the human heavy and light chainloci, PCR analysis was performed on genomic DNA from the liver of 58 dayfetus #5580, 57 day fetus # 5848, and 91 day fetuses #5442A and 5442B.The PCR primers used for detection of the heavy chain loci were VH3-F(5′-AGTGAGATAAGCAGTGGATG-3′, SEQ ID NO: 18) and VH3-R(5′-CTTGTGCTACTCCCATCACT-3′, SEQ ID NO: 19), and the primers used forthe detection of the light chain were IgL-F(5′-GGAGACCACCAAACCCTCCAAA-3′, SEQ ID NO: 20) and IgL-R(5′-GAGAGTTGCAGAAGGGGTYGACT-3′, SEQ ID NO: 21). The PCR reactionmixtures contained 18.9 μl water, 3 ul of 10×Ex Taq buffer, 4.8 μl ofdNTP mixture, 10 pmol forward primer, 10 pmol of reverse primer, 1 μl ofgenomic DNA, and 0.3 ul of Ex Taq. Thirty-eight PCR cycles wereperformed as follows: 85° C. for three minutes, 94° C. for one minute,98° C. for 10 seconds, 56° C. for 30 seconds, and 72° C. for 30 seconds(FIGS. 13 and 14).

As illustrated in FIGS. 13 and 14, positive control 58 day fetus #5580contained both human heavy and light chain immunoglobulin loci.Additionally, the 91 day fetuses #5442A and 5442B also contained bothheavy and light chain loci (FIG. 14). In contrast, fetus #5848 did notcontain either human loci and may not have contained ΔΔHAC. Theseresults suggested that ΔΔHAC can be stably retained up to gestationalday 91 in bovine.

Rearrangement and Expression of Human Heavy Chain Locus in ΔΔHAC Fetus#5442A RT-PCR was used to detect expression of rearranged human heavychain RNA transcripts in ΔΔHAC fetus #5542A. The RT-PCR primers usedwere CH3-F3 (5′-GGAGACCACCAAACCCTCCAAA-3′, SEQ ID NO: 22) and CH4-R2(5′-GAGAGTTGCAGAAGGGGTGACT-3′, SEQ ID NO: 23). The RT-PCR reactionmixtures contained 18.9 μl water, 3 μl of 10×Ex Taq buffer, 4.8 μl ofdNTP mixture, 10 pmol forward primer, 10 pmol of reverse primer, 1 μl ofcDNA, and 0.3 μl of Ex Taq. Forty cycles of RT-PCR cycles were performedas follows: 85° C. for three minutes, 94° C. for one minute, 98° C. for10 seconds, 60° C. for 30 seconds, and 72° C. for 30 seconds.

Lanes 4 and 5 of FIG. 15 contained amplified spliced mu heavy chainconstant region sequences from the spleen of fetus #5442A that aresimilar in size to that of the positive control samples. These resultsindicate that fetus #5442A expressed a rearranged mu heavy chaintranscript in its spleen. Faint bands were also seen in the region ofthe unspliced genomic sequence, which are amplified from genomic DNAcontaminated in the RNA sample. Control samples from the liver and brainof fetus #5442A did not produce a band of the size expected for anamplified rearranged heavy chain sequence.

Rearrangement and Expression of Human Heavy Chain Locus in ΔΔHAC Fetus#5868A RT-PCR was used to detect expression of rearranged human heavychain RNA transcripts in the spleen of a ΔΔHAC fetus at 119 gestationaldays (fetus #5868A). The primers used for this analysis were VH30-3(5′-caggtgcagctggtggagtctgg-3′, SEQ ID NO: 24) and CM-1(5′-caggagaaagtgatggagtc-3′, SEQ ID NO: 25). Additionally, primers“GAPDH up” (5′-gtcatcatctctgccccttctg-3′, SEQ ID NO: 26) and “GAPDHdown” (5′-aacaacttcttgatgtcatcat-3′, SEQ ID NO: 27) were used to amplifyGAPDH control transcripts. For this PCR analysis, the reaction mixturewas incubated at 95° C. for five minutes and then multiple cycles ofdenaturation, annealing, and amplification were performed by incubationat 95° C. for one minute, 58° C. for one minute, and 72° C. for twominutes. Then, the mixture was incubated at 72° C. for 10 minutes.

Lane 3 of FIG. 16 contains the RT-PCR product produced from thisanalysis of ΔΔHAC fetus# 5868A. This RT-PCR product was the sizeexpected for the amplification of a rearranged human heavy chain (470base pairs) and migrated to the same position in the gel as the controlcDNA known to contain sequences corresponding to rearranged human heavychain transcripts. As controls, both ΔΔHAC fetus# 5868A fetal spleencDNA and normal bovine cDNA samples generated a product when amplifiedwith GAPDH primers, demonstrating the capacity of the cDNA to supportamplification (lanes 7 and 8).

Rearrangement and Expression of Human Lambda Locus in ΔΔHAC Fetuses#5442A and 5442B Primers specific for amplification of a transcriptincluding portions of human lambda were used to detect RNA transcriptsfrom a rearranged human lambda light chain locus.

For the RT-PCR analysis shown in FIG. 17, an equimolar mixture ofprimers Cλ1 (.5′-GGGAATTCGGGTAGAAGTTCACTGATCAG-3′, SEQ ID NO: 28), Cλ2-3(5′-GGGAATTCGGGTAGAAGTCACTTATGAG-3′, SEQ ID NO: 29), and Cλ7(5′-GGGAATTCGGGTAGAAGTCACTTACGAG-3′, SEQ ID NO: 30) was used with primerVλ1 LEA1 (5′-CCCCCAAGCTTRCCKGSTYYCCTCTCCTC-3′, SEQ ID NO: 31). TheRT-PCR reaction mixtures contained 18.9 μl water, 3 μl of 10×Ex Taqbuffer, 4.8 μl of dNTP mixture, 10 pmol forward primer, 10 pmol ofreverse primer, 1 μl of cDNA and 0.3 μl of Ex Taq. The RT-PCR conditionswere as follows: 40 cycles of 85° C. for three minutes, 94° C. for oneminute, 98° C. for 10 seconds, 60° C. for 30 seconds, and 72° C. for oneminute.

As shown in FIG. 18, this RT-PCR analysis was also performed using anequimolar mixture of primers Vλ3LEA1(5′-CCCCCAAGCTTGCCTGGACCCCTCTCTGG-3′; SEQ ID NO:32), Vλ3JLEAD(5′-ATCGGCAAAGCTTGGACCCCTCTCTGGCTCAC-3′, SEQ ID NO: 33), VλBACK4(5′-CCCCCAAGCTTCTCGGCGTCCTTGCTTAC-3′, SEQ ID NO: 34) and an equimolarmixture of primers Cλ1 (5′-GGGAATTCGGGTAGAAGTTCACTGATCAG-3′, SEQ ID NO:35) Cλ2-3 (5′-GGGAATTCGGGTAGAAGTCACTTATGAG-3′, SEQ ID NO: 36) and Cλ7(5′-GGGAATTCGGGTAGAAGTCACTTACGAG-3′, SEQ ID NO: 37). The RT-PCR reactionconditions were the same as those described above for FIG. 7.

Lanes 6 and 7 of FIG. 17 and lanes 4 and 5 of FIG. 18 contained RT-PCRproducts from the spleen of fetus #5442A that are similar in size to thepositive control bands, indicating the presence of rearranged lightchain RNA transcripts in this fetus. The spleen sample from fetus #5442Bproduced very weak bands of the appropriate size which are not visiblein the picture. This RT-PCR product indicates that fetus #5442B alsoexpressed a rearranged light chain immunoglobulin transcript in itsspleen. As expected, samples from the brain of fetuses #5442A and 5442Bdid not express human rearranged lambda light chain transcripts.

Rearrangement and Expression of Human Lambda Locus in ΔΔHAC Fetus #5868ARNA transcripts from a rearranged human lambda light chain locus werealso detected in ΔΔHAC fetus# 5868A. For this analysis, primers specificfor amplification of a transcript including portions of human lambdawere used to detect ΔΔHAC-encoded expression of transcripts encodingportions of a rearranged human lambda locus. Primer VL1 LEAI(5′-cccccaagcttRccKgStYYcctctcctc-3′; SEQ ID NO:38) and an equimolarmixture of primers CL1 (5′-gggaattcgggtagaagtcactgatcag-3′; SEQ IDNO:39), CL2-3 (5′-gggaattcgggtagaagtcacttatgag-3′; SEQ ID NO:40), andCL7 (5′-gggaattcgggtagaagtcacttacgag-3′; SEQ ID NO:41) were used forthis analysis. For this RT-PCR reaction, the reaction mixtures wereincubated at 95° C. for 5 minutes and then multiple cycles ofdenaturation, annealing, and amplification were performed by incubationat 95° C. for one minute, 60° C. for one minute, and 72° C. for twominutes. Then, the mixtures were incubated at 72° C. for 10 minutes.

This analysis demonstrated that spleen cDNA from ΔΔHAC #5868A (lane 4 ofFIG. 19) produced a RT-PCR product of the same size as the TC mousespleen cDNA (lane 6) positive control. No such RT-PCR product wasdetected using either brain or liver cDNA from ΔΔHAC #5868A (lanes 2 and3, respectively). The capacity of each of these tissues to supportRT-PCR was shown by successful amplification of the housekeeping gene,GAPDH using primers “GAPDH up” and “GAPDH down” (lanes 8 and 10).

Verification of ΔΔHAC Rearrangement by Sequencing RT-PCR analysis wasperformed on a spleen sample from fetus #5442A using an equimolarmixture of primers Cλ1, Cλ2-3, and Cλ7 with primer Vλ1LEA1, or anequimolar mixture of primersVλ3LEA1, Vλ3JLEAD, and VλBACK4 and anequimolar mixture of primers Cλ1, C/2-3, and Cλ7 in. The PCR productswere purified using a CHROMA SPIN column (CLONETECH) and cloned into thepCR2.1 TA-cloning vector (Invitrogen), according to manufacturer'sprotocol. The Dye Terminator sequence reaction (ABI Applied System) wascarried out using the Cλ1, Cλ2-3, and Cλ7 primers in an equimolarmixture. Twenty-five cycles were performed at 96° C. for one minute, 96°C. for 10 seconds, 55° C. for five seconds, and 60° C. for four minutes.The 10 μl reaction mixture contained BigDye Terminator reaction mixture(3 μl), template plasmid (200 ng), and the Cλ1, Cλ2-3, and Cλ7 primers(1.6 pmol). The reaction mixture was analyzed using a ABI 3700sequencer.

At least two rearranged human lambda light chain transcripts wereidentified (V1-17/JL3/Cλ and V2-13/JL2/Cλ). These results demonstratethat VJ rearrangement of human lambda light chain genes occurs in theΔΔHAC in the spleen of fetus #5442A (FIGS. 20 and 21).

FACS Analysis of Expression of Human Lambda Light Chain and Bovine HeavyChain in ΔΔHAC Fetus #5442A and 5442B Splenic lymphocytes from ΔΔHACFetus #5442A and 5442B were analyzed for the expression of human lambdalight chain and bovine heavy chain proteins. These cells were reactedwith a phycoerytherin labeled anti-human lambda antibody (FIGS. 22C and22D), a FITC labeled anti-bovine IgM antibody (FIGS. 22D and 22H), or noantibody (FIGS. 22A, 22B, 22E, and 22F) for 20 minutes at 4° C. Cellswere then washed twice with PBS plus 2% FCS and analyzed on a FASCaliburcell sorter. The percent of cells reacting with the antibody wascalculated using the non antibody controls to electronically se thegates. These percentages are displayed beneath each histogram. Fetus #5442A (FIGS. 22A–22D) and fetus #5442B (FIGS. 22E–22H) expressed bothhuman lambda light chain protein and bovine heavy chain protein.

EXAMPLE 3 Transgenic Ungulates Producing Xenogenous Antibodies andReduced Amounts of Endogenous Antibodies

Transgenic ungulates expressing a xenogenous antibody and having areduced level of expression of endogenous antibodies may also begenerated. By increasing the number of functional xenogenousimmunoglobulin heavy or light chain genes relative to the number offunctional endogenous heavy or light chain genes, the percentage of Bcells expressing xenogenous antibodies should increase.

To generate these transgenic ungulates, ΔHAC or ΔΔHAC transgenicungulates may be mated with transgenic ungulates containing a mutationin one or both alleles of an endogenous immunoglobulin chain (e.g., a muheavy chain or a lambda or kappa light chain). If desired, the resultingtransgenic ungulates may be mated with (i) transgenic ungulatescontaining a mutation in one or both alleles of an endogenousalpha-(1,3)-galactosyltransferase, prion, and/or J chain nucleic acid or(ii) transgenic ungulates containing an exogenous J chain nucleic acid(e.g., human J chain). Alternatively, a cell (e.g., a fetal fibroblast)from a ΔHAC or ΔΔHAC transgenic fetus may be genetically modified by themutation of one or more endogenous immunoglobulin genes. In anotherpossible method, ΔHAC or ΔΔHAC is introduced into a cell (e.g., a fetalfibroblast) in which endogenous immunoglobulins (mu heavy and/or lambdalight chains) are hemizgously or homozygously inactivated. In any of theabove methods, the cells may also be genetically modified by (i) theintroduction of a mutation, preferably a knockout mutation, into one orboth alleles of an endogenous alpha-(1,3)-galactosyltransferase, prion,and/or J chain nucleic acid or (ii) the introduction of an exogenous Jchain nucleic acid. The resulting transgenic cell may then be used innuclear transfer procedures to generate the desired transgenicungulates. Exemplary methods are described below.

DNA Constructs The mu heavy chain (FIG. 2A), lambda light chain, kappalight chain, alpha-(1,3)-galactosyltransferase, prion, and/or J chainknockout constructs described above may be used. Alternatively, thepuromycin resistant mu heavy chain construct described below may be used(FIG. 3F). This knockout construct was designed to remove the 4 maincoding exons of the bovine mu heavy chain locus but leave thetransmembrane domain intact, resulting in the inactivation of the muheavy chain locus.

The puromycin resistant construct was assembled as follows. A 4.4kilobase XhoI fragment containing the region immediately proximal tocoding exon 1 was inserted into the XhoI site of pBluescript II SK+.Plasmid pPGKPuro, which contains a puromycin resistant gene, wasobtained from Dr. Peter W. Laird, Whitehead Institute, USA. A 1.7 KbXhoI fragment containing a puromycin resistance gene was subclonedadjacent to, and downstream of, the 4.4 Kb fragment into the SalI sitepresent in the polylinker region. This 1.7 Kb puromycin marker replacesthe coding exons CH1, CH2, CH3 and CH4 of the bovine immunoglobulinheavy chain locus. An XbaI fragment containing a 4.6 Kb region of the mulocus that is downstream of these four exons in the wild-type genomicsequence was added to this construct for use as the second region ofhomology.

To generate the final targeting construct, a subclone of this constructwas generated by cutting the three assembled fragments with NotI andMluI The MluI restriction digestion truncates the 4.6 Kb fragment downto 1.4 Kb. The NotI site lies in the polylinker and does not cut intothe subcloned DNA itself. The MluI site was filled in with a Klenowfragment to generate a blunt end, and the NotI/filled in MluI fragmentwas subcloned into a fresh pBluescript II SK+ vector using the NotI andSmal sites present in the pBluescript vector. For gene targeting, thefinal vector is linearized with NotI.

Gene Targeting by Electroporation and Drug Selection of TransfectedFibroblasts For electroporation, a single cell suspension of 1×10⁷bovine fetal fibroblasts (e.g, fibroblasts obtained as described inExample 2 from a ΔHAC or ΔΔHAC transgenic fetus) that had undergone alimited number of population doublings is centrifuged at 1200 rpm forfive minutes and re-suspended in 0.8 ml of serum-free Alpha-MEM medium.The re-suspended cells are transferred to a 0.4 cm electroporationcuvette (Invitrogen, cat#. P460-50). Next, 30 μg of a restrictionenzyme-linearized, gene targeting vector DNA is added, and the contentsof the cuvette are mixed using a 1 ml pipette, followed by a two minuteincubation step at room temperature. The cuvette is inserted into theshocking chamber of a Gene Pulser II electroporation system (Biorad) andthen electroporated at 1000 volts and 50 μF. The cuvette is quicklytransferred to a tissue culture hood and the electroporated cells arepipetted into approximately 30 ml of complete fibroblast medium. Thecells are equally distributed into thirty 100 mm tissue culture dishes(Corning, cat#. 431079), gently swirled to evenly distribute the cells,and incubated at 38.5° C./5% CO₂ for 16 to 24 hours The media is removedby aspiration and replaced with complete fibroblast medium containingthe selection drug of choice. The media is changed every two days andcontinued for a total time period of 7 to 14 days. During the drugselection process, representative plates are visually monitored to checkfor cell death and colony formation. Negative control plates are set upthat contained fibroblasts that are electroporated in the absence of thegene targeting vector and should yield no colonies during the drugselection process.

Picking of Drug Resistant Fibroblast Colonies and Expansion of CellsFollowing completion of the drug selection step (usually 7 to 14 days),the drug resistant colonies are macroscopically visible and ready fortransfer to 48 well tissue culture plates for expansion. To assist inthe transferring process, individual colonies are circled on the bottomof the tissue culture plate using a colored marker (Sharpie). Tissueculture plates containing colonies are washed 2× with 1×D-PBS (withoutCa²⁺ and Mg²⁺) and then 5 ml of a 1:5 dilution of the cell dissociationbuffer is added per plates. Following a 3 to five minute roomtemperature incubation step, individual colonies start to detach fromthe bottom of the tissue culture dish. Before the colonies detached,they are individually transferred to a single well of a 48 well tissueculture plate using a P200 pipetmen and an aerosol barrier pipette tip(200 or 250 μl). Following transfer, the colony is completelydissociated by pipeting up-and-down and 1 ml of complete fibroblastmedium is added. To ensure that the cells are drug resistant, drugselection is continued throughout the 48 well stage. The transferredcolonies are cultured at 38.5° C./5% CO₂ and visually monitored using aninverted microscope. Two to seven days later, wells that are approachingconfluency are washed two times with 1×D-PBS (without Ca²⁺ and Mg²⁺) anddetached from the bottom of the well by the addition of 0.2 ml of celldissociation buffer, followed by a five minutes room temperatureincubation step. Following detachment, the cells are further dissociatedby pipeting up-and-down using a P1000 pipetmen and an aerosol pipettetip (1000 μl). Approximately 75% of the dissociated fibroblasts aretransferred to an individual well of a 24 well tissue culture plate toexpand further for subsequent PCR analysis and the remaining 25% istransferred to a single well of a second 24 well plate for expansion andeventually used for somatic cell nuclear transfer experiments. Whencells in the plate containing 75% of the original cells expanded to nearconfluency, DNA is isolated from that clone for genetic analysis.

DNA Preparation The procedure used to isolate DNA for genetic analysesis adapted from Laird et al, Nucleic Acids Research, 1991, Volume 19,No. 15. In particular, once a particular clone has attainednear-confluency in one well of a 24 well plate, culture medium isaspirated from that well and the adherent cells are washed twice withPBS. The PBS is aspirated off and replaced with 0.2 ml buffer to lysethe cells and digest excess protein from the DNA to be isolated. Thisbuffer is composed of 100 mM Tris-HCl (pH 8.5), 5 mM EDTA, 0.2% SDS, 200mM NaCl and 100 ug/ml proteinase K. The 24 well plate is returned to thetissue culture incubator for a minimum of three hours to allow therelease of the DNA and digestion of protein. The viscous product of thisprocedure is transferred to a 1.5 ml microcentrifuge tube and 0.2 ml ofisopropanol added to precipitate the DNA. The precipitate is recoveredby centrifugation, the DNA pellet is rinsed with 70% ethanol, and afterair-drying, the pellet is resuspended in 25–50 ul of buffer containing10 mM Tris, pH 8, and 1 mM EDTA. This DNA is used for PCR analyses ofclones.

Screening of Clones Two different approaches are used to screen clones,both employing the polymerase chain reaction (PCR). All approachesdescribed in this section are adaptable to the targeting of any othergene, the only difference being the sequences of the primers used forgenetic analysis.

According to the first approach, two separate pairs of primers are usedto independently amplify products of stable transfection. One pair ofprimers is used to detect the presence of the targeting vector in thegenome of a clone, regardless of the site of integration. The primersare designed to anneal to DNA sequences both present in the targetingvector. The intensity of the PCR product from this PCR reaction may becorrelated with the number of copies of the targeting vector that haveintegrated into the genome. Thus, cells containing only one copy of thetargeting vector tend to result in less intense bands from the PCRreaction. The other pair of primers is designed to detect only thosecopies of the vector that integrated at the desired locus. In this case,one primer is designed to anneal within the targeting vector and theother is designed to anneal to sequences specific to the locus beingtargeted, which are not present in the targeting vector. In this case, aPCR product is only detected if the targeting vector has integrateddirectly next to the site not present in the targeting vector,indicating a desired targeting event. If product is detected, the cloneis used for nuclear transfer.

For the neomycin resistant heavy chain knockout construct, primers Neol(5′-CTT GAA GAC GAA AGG GCC TCG TGA TAC GCC-3′, SEQ ID NO: 42) andIN2521 (5′-CTG AGA CTT CCT TTC ACC CTC CAG GCA CCG-3′, SEQ ID NO: 43)are used to detect the presence of the targeting vector in cells,regardless of the location of integration. Primers Neol and OUT3570(5′-CGA TGA ATG CCC CAT TTC ACC CAA GTC TGT C-3′, SEQ ID NO: 44) areused to specifically amplify only those copies of the targetingconstruct that integrated into the mu heavy chain locus.

For these PCR reactions to analyze the integration of the neomycinresistant heavy chain knockout construct, a Qiagen PCR kit is used. ThePCR reaction mixture contains 1 pmole of each primer, 5 ul of 10×reaction buffer, 10 ul of Q solution, 5 ul of DNA, and 1 ul of dNTPsolution. The reaction mixture is brought to a total volume of 50 ulwith H₂O. This PCR amplification is performed using an initialdenaturing incubation at 94° C. for two minutes. Then, 30 cycles ofdenaturation, annealing, and amplification are performed by incubationat 94° C. for 45 seconds, 60° C. for 45 seconds, and 72° C. for twominutes. Then, the reaction mixture is incubated at 72° C. for fiveminutes and at 4° C. until the mixture is removed from the PCR machine.

In the alternative approach, a single primer set is used to amplify thetargeted locus and the size of the PCR products is diagnostic forcorrect targeting. One primer is designed to anneal to a region of thelocus not present in the targeting vector and the other primer isdesigned to anneal to a site present in the targeting vector but alsopresent in the wild type locus. In this case, there is no detection oftargeting vector that had integrated at undesirable sites in the genome.Because the region deleted by the targeting vector is different in sizefrom the drug selection marker inserted in its place, the size of theproduct depended on whether the locus amplified is of wild-type genotypeor of targeted genotype. Amplification of DNA from clones containingincorrect insertions or no insertions at all of the targeting vectorresults in a single PCR product of expected size for the wild typelocus. Amplification of DNA from clones containing a correctly targeted(“knocked out”) allele results in two PCR products, one representingamplification of the wild type allele and one of altered, predictablesize due to the replacement of some sequence in the wild-type allelewith the drug resistance marker, which is of different length from thesequence it replaced.

For the puromycin resistant heavy chain knockout construct, primersShortend (5′-CTG AGC CAA GCA GTG GCC CCG AG-3′, SEQ ID NO: 45) andLongend (5′-GGG CTG AGA CTG GGT GAA CAG AAG GG-3′, SEQ ID NO: 46) areused. This pair of primers amplifies both the wild-type heavy chainlocus and loci that have been appropriately targeted by the puromycinconstruct. The size difference between the two bands is approximately0.7 Kb. The presence of the shorter band is indicative of appropriatetargeting.

For this PCR reaction to analyze the integration of the puromcyingresistant heavy chain knockout construct, a Promega Master Mix kit isused. The PCR reaction mixture contains 1 pmole of each primer, 2.5 ulof DNA, and 25 ul of 2× Promega Master Mix. The reaction mixture isbrought to a total volume of 50 ul with H₂O. This PCR amplification isperformed using an initial denaturing incubation at 94° C. for twominutes. Then, 30 cycles of denaturation, annealing, and amplificationare performed by incubation at 94° C. for 45 seconds, 60° C. for 45seconds, and 72° C. for two minutes. Then, the reaction mixture isincubated at 72° C. for five minutes and at 4° C. until the mixture isremoved from the PCR machine.

First Round of Nuclear Transfer Selected fibroblast cells in which animmunoglobulin gene has been inactivated may be used for nucleartransfer as described in Example 2 to generate a transgenic ungulatecontaining a mutation in an endogenous immunoglobulin gene andcontaining a HAC encoding an xenogenous immunoglobulin gene.Alternatively, nuclear transfer may be performed using standard methodsto insert a nucleus or chromatin mass (i.e., one or more chromosomes notenclosed by a membrane) from a selected transgenic fibroblast into anenucleated oocyte (U.S. Ser. No. 60,258,151; filed Dec. 22, 2000). Thesemethods may also be used for cells in which an endogenousalpha-(1,3)-galactosyltransferase, prion, and/or J chain nucleic acidhas been mutated.

Second Round of Mutagenesis and Nuclear Transfer If desired, a cell(e.g., a fetal fibroblast) may be obtained from a transgenic ungulategenerated from the first round of nuclear transfer. Another round ofgene targeting may be performed as described above to inactivate thesecond allele of the gene inactivated in the first round of targeting.Alternatively, another immunoglobulin (e.g., mu heavy chain, lambdalight chain, kappa light chain, or J chain),alpha-(1,3)-galactosyltransferase, or prion gene may be inactivated inthis round of targeting. For this second round of targeting, either ahigher concentration of antibiotic may be used or a knockout constructwith a different antibiotic resistance marker may be used. Antibioticresistance cells may be selected as described above. The selected cellsmay be used in a second round of nuclear transfer as described above togenerate, for example, a transgenic ungulate containing two mutations inendogenous immunoglobulin genes and containing a HAC encoding anxenogenous immunoglobulin gene. Alternatively, the selected antibioticresistant cells may first be treated to isolate G1 phase cells asdescribed below, which are used for the second round of nucleartransfer.

To isolation of G1 cells for nuclear transfer, 5.0×10⁵ cells are platedonto 100 mm tissue culture plates containing 10 ml of α-MEM+FCS, twentyfour hours prior to isolation. The following day, plates are washed withPBS and the culture medium is replaced for 1–2 hours before isolation.The plates are then shaken for 30–60 seconds on a Vortex-Genie 2 (FisherScientific, Houston, Tex., medium speed), the medium is removed, spun at1000 G for five minutes and the pellet is re-suspended in 250 μl of MEM+FCS. Newly divided cell doublets attached by a cytoplasmic bridge, arethen selected, as these cells are in early G1. This isolation procedureis referred to as the “shake off” method.

EXAMPLE 4 Transgenic Ungulates Having Reducedα-1,3-galactosyltransferase Activity

Bovine fibroblast cell lines in which one allele of theα-1,3-galactosyltransferase locus is mutated were generated byhomologous recombination. The DNA construct for generating theα-galactosyltransferase knockout cells was used to prevent transcriptionof functional, full-length α-galactosyltransferase mRNA by insertingboth a puromycin-resistance gene (puro, described in Example 3) and atranscription termination cassette (STOP) in exon 9 which contains thecatalytic domain. Thus, the resulting immature α-galactosyltransferasetranscripts lack the catalytic domain. The DNA construct (i.e., theα-galactosyltransferase KO vector) was electroporated into threeindependent bovine fibroblast cell lines, and then puromycin-resistantcolonies were isolated. Based on PCR analysis, homologous recombinationin the exon 9 region occurred in some colonies. Thus, bovine fibroblastcell lines in which one allele of α1,3-galactosyltransferase locus ismutated were generated. If desired, the second allele can be mutated byusing the same knockout vector and a higher concentration of antibioticto select for homozygous knockout cells or using another knockout vectorwith a different antibiotic resistance gene. This method may also beapplied to cells from other ungulates to generate transgenic cells foruse in the nuclear transfer methods described herein to producetransgenic ungulates of the present invention.

These methods are described further below.

Construction of an α-1,3-galactosyltransferase KO Vector Theα-1,3-galactosyltransferase KO vector was generated as follows (FIG.23). To isolate genomic DNA around exon 9 of theα-1,3-galactosyltransferase gene, a DNA probe was amplified by PCR usingthe following primer pair 5′-gatgatgtctccaggatgcc-3′ (SEQ ID NO: 61) and5′-gacaagcttaatatccgcagg-3′ (SEQ ID NO: 62). Using this probe, a bovinegenomic λ phage library was screened, and 7 positive λ phage clones wereidentified. One clone, which contained DNA from a male Charolais bovinefibrolast cell, was analyzed further by restriction mapping. The NotI-Xho I genomic fragment containing exon 9 was subcloned intopBluescript II SK(−), and then both puro and STOP cassettes wereinserted at the Avi I site in the Not I-Xho I genomic fragment which is5′ to the catalytic domain. A diphtheria toxin gene (DT-A, Gibco) wasalso added to the vector construct to kill cells in which the targetingcassette was integrated nonhomologously.

Transfection/Knockout Procedures Transfection of three fetal fibroblastscell lines (two from a male Jersey bovine and one from a female Jerseybovine) was performed using a standard electroporation protocol asfollows. The medium used to culture the bovine fetal fibroblastscontained 500 ml Alpha MEM (Gibco, 12561-049), 50 ml fetal calf serum(Hy-Clone #ABL13080), 5 ml penicillin-streptomycin (SIGMA), and 1 ml2-mercaptoethanol (Gibco/BRL #21985-023). On the day prior totransfection, cells were seeded on a T175 tissue culture flask with atargeted confluency of 80–100%, as determined by microscopicexamination. On the day of transfection, about 10⁷ bovine fibroblastscells were trypsinized and washed once with alpha-MEM medium. Afterresuspension of the cells in 800 μl of alpha-MEM, 30 μg of DNA was addedto the cell suspension and mixed well by pipetting. The cell-DNAsuspension was transferred into an electroporation cuvette andelectroporated at 1,000 V and 50 μF. After that, the electroporatedcells were plated onto twenty 24-well plates with the alpha-MEM mediumsupplemented with the serum. After a 48 hour-culture, the medium wasreplaced with medium containing 1 μg/ml of puromycin, and the cells werecultured for 2–3 weeks to select puromycin resistant cells. Afterselection, all colonies which reached close to 100% confluency werepicked, and genomic DNA was extracted from the colonies to screen forthe desired homologous recombination events by PCR.

Screening for Targeted Integrations As described above, the genomic DNAwas extracted from each 24-well independently using the PUREGENE DNAisolation Kit (Gentra SYSTEMS) according to the manufacture's protocol.Each genomic DNA sample was resuspended in 20 μl of 10 mM Tris-Cl (pH8.0) and 1 mM EDTA (EDTA). Screening by PCR was performed using thefollowing primer pair 5′-aagaagagaaaggtagaagaccccaaggac-3′ (SEQ ID NO:63) and 5′-cctgggtatagacaggtgggtattgtgc-3′ (SEQ ID NO: 64). The sequenceof one primer is located in the α-1,3-galactosyltransferase KO vector,and the sequence of the other primer is located just outside of theintegrated vector in the targeted endogenous locus (FIG. 23). Therefore,the expected PCR product should be detected only when the KO vector isintegrated into the targeted locus by homologous recombination.

The PCR reaction mixtures contained 18.9 μl water, 3 μl of 10×LA PCRbuffer II (Mg²⁺ plus), 4.8 μl of dNTP mixture, 10 pmol forward primer,10 pmol of reverse primer, 1 μl of genomic DNA, and 0.3 μl of LA Taq.Forty cycles of PCR were performed by incubating the reaction mixturesat the following conditions: 85° C. for three minutes, 94° C. for oneminute, 98° C. for 10 seconds, and 68° C. for 15 minutes. After PCR, thereaction mixtures were analyzed by electrophoresis. Puromycin-resistantclones which generated PCR products of the expected size were selected(FIG. 23). Thus, bovine fibroblast cell lines in which one allele of theα-1,3-galactosyltransferase locus is mutated by the KO vector weresuccessfully generated.

EXAMPLE 5

Alternative Method for Producing Transgenic Ungulates usingAdeno-Associated Viruses to Mutate an Endogenous Gene

Adeno-associated virus (AAV) can be used for specific replacement oftargeted sequences present in the genome of cells (Inoue et al., Mol.Ther. 3(4):526–530, 2001); Hirata et al., J Virol. 74(10):16536–42,2000); Inoue et al., J. Virol. 73(9):7376–80, 1999); and Russell et al.,Nat. Genet. 18(4):325–30,1998)). The gene targeting rate is highlyefficient in comparison to more conventional gene targeting approaches.AAV has a broad range of host and tissue specificities, includingspecificity for both bovine and human skin fibroblasts. Thus, AAV can beused to produce transgenic ungulate cells containing one or moremutations in an endogenous immunoglobulin (e.g., mu heavy chain, lambdalight chain, kappa light chain, or J chain),alpha-(1,3)-galactosyltransferase, or prion gene. These transgenic cellscan then be used in the nuclear transfer methods described herein toproduce transgenic ungulates of the present invention.

Using AAV resulted in homologous recombination of the bovineimmunoglobulin heavy chain locus at higher frequencies than previouslyobtained using traditional gene targeting strategies (i.e.,electroporation and lipofection procedures). In the first round of genetargeting experiments, five appropriately targeted fibroblast cloneswere obtained out of 73 stable transductants containing the DNAintroduced through an AAV vector.

These experiments were carried out as follows.

AAV Knockout Vectors AAV constructs can disrupt a gene either by simpleinsertion of foreign sequences or replacement of endogenous sequenceswith new sequence present in the AAV vector. FIG. 24 shows an AAVconstruct in which all four coding exons of the bovine immunoglobulinheavy chain mu constant region are present on a 2822 base pairBamHI-XhoI fragment. A 1.16 Kb fragment containing a neomycin resistancemarker present in the commercially available vector, pMC1Neo, wasinserted into a SacII site present in exon 4 of the mu heavy chain locusfrom a Holstein bovine. This locus is the one contained in the phageclone isolated to generate the knockout vector described in Example 1.To generate the AVV vector, the SacII site in the mu heavy chain locuswas filled in to create blunt ends, which were then ligated to bluntSalI linkers (New England Biolabs). Then, the XhoI fragment of pMC1Neo,which contains the neomycin resistance gene, was ligated to the SalIsite added to the locus through the Sail linker. This ligation can beperformed because the XhoI and SalI restriction sites have compatibleends. This knockout vector causes a disruptional insertion of theneomycin resistance gene into the endogenous mu heavy chain gene,thereby inactivating the mu heavy chain gene. This gene inactivationoccurs without deleting regions of the endogenous mu locus.

An alternative vector was designed to remove exons 3 and 4 from theendogenous locus during targeting, resulting in the replacement of thesetwo exons with a functional copy of the neomycin resistance gene (FIG.25). This construct was generated using PCR amplification of genomic DNAfrom a female Jersey bovine. In particular, the 3′ region of homologywas amplified using the following primers: 5′GGGGTCTAGAgcagacactacactgatgggcccttggtcc 3′ (SEQ ID NO: 65), which addsa XbaI restriction site, and 5′ GGGGAAGCTTcgtgtccctggtcctgtctgacacag 3′(SEQ ID NO: 66), which adds a HindIII restriction site. The 5′ region ofhomology was amplified with primers 5′GGGGCTCGAGgtcggcgaaggatggggggaggtg 3′ (SEQ ID NO: 67), which adds a XhoIrestriction site, and 5′ GGGGGGTACCgctgggctgagctgggcagagtggg 3′ (SEQ IDNO: 68), which adds a KpnI restriction site. The capitalized nucleotidesin these primer sequences are nucleotides that do not anneal to the muheavy chain locus but are included in the primers to add restrictionsites to facilitate later subcloning steps. The first four guanines areadded to separate the restriction sites from the very end of the primersbecause restriction enzymes do not cleave sites that are at the very endof primers as well as internal sites. The 5′ region of homology is 1.5Kb long and contains exons 1 and 2. The 5′ region of homology alsocontains the first 25 nucleotides of exon 3 to maintain the spliceacceptor site of exon 3. The splice acceptor site allows exon 3 to beused for splicing and thus prevents the possible splicing of exons 1 and2 to the downstream transmembrane domain to form an aberrantmembrane-bound product. The 3′ region of homology is 1.24 Kb long andcontains the region immediately downstream of exon 4.

For the construct shown in FIG. 24, the targeting cassette was insertedinto the AAV vector reported by Ryan et al. (J. of Virology70:1542–1553, 1996), which contains viral long terminal repeat (LTR)sequences, using standard methods. The AAV vector was packaged intocapsids using the TtetA2 packaging cell line as previously described(Inoue and Russell, 1998, J. Virol. 72:7024–7031, 1998) and purified aspreviously described (Zolotukhin et al., Gene Therapy, 6: 973–985,1999). For the construct shown in FIG. 25, the above method or any otherstandard method can be used to insert the targeting cassette into theAAV vector described by Ryan et aL or any other AAV vector (such as acommercially available vector from Stratagene) and generate virusescontaining the vector.

Transduction Procedures Fibroblasts from a female Jersey bovine wereseeded into one well of a 48 well tissue culture plate at 40,000 cellsper well and cultured in complete medium at 38.5° C. and 5% CO₂ untilcells attached to the bottom surface of the well. Once cells adhered,the medium was removed and replaced with 0.2 ml of fresh mediumcontaining AAV particles with the vector shown in FIG. 24 at amultiplicity of infection (MOI) of 500–20,000 particles/cell. The MOIwas chosen based on pilot experiments that determined the resultingnumbers of colonies and the spacing of the colonies during the drugselection phase. Plates were incubated overnight. After this incubation,the transduced wells were rinsed with calcium and magnesium-free PBS anddetached from the wells using either trypsin or the cell dissociationbuffer described above. A uniform cell suspension was obtained by gentlepipetting of the detached cells, and the cells from the well wereredistributed among ten 100 mm tissue culture dishes. Dishes wereincubated with complete medium overnight.

Following this incubation of the 100 mm dishes, the medium was replacedwith selective medium containing G418 at a concentration of 350micrograms/ml. Selective medium was changed every 2–3 days untilcolonies were macroscopically visible on the surface of the dish. Atthat point, individual colonies were picked and transferred into theirown vessels.

Regions containing colonies were marked on the outer surface of thetissue culture dish. Once all colonies were circled, medium wasaspirated off the plates, and the plates were washed three times withcalcium and magnesium-free PBS. After washing, the plates were floodedwith a 1:25 dilution of 1× trypsin and allowed to sit at roomtemperature until the colonies had visibly begun to detach from thesurface of the plate. Plates were kept stationary to prevent detachedcolonies from floating to another location of the plate. A pipet tip wasused to pick up cell clumps in a volume of 50 microliters, and thecontents of the pipet tip were transferred into one well of a 24 welltissue culture plate. Once all colonies were transferred, completemedium containing G418 was added, and the isolated clones were allowedto proliferate to near confluency.

When an individual well was close to confluency, it was washed twicewith calcium and magnesium free PBS. Cells were detached using 0.2 ml ofcell dissociation buffer. Of this cell suspension, 20 μl was transferredto a new 24 well plate, and the remaining cells were allowed to reattachto the surface of the original 24 well plate following the addition of2.0 ml of complete medium. The original plate was incubated to 100%confluency. The new plate serves as a source of appropriately targetedcells for future bovine cloning procedures.

When a well from the original 24 well plate became 100% confluent, themedium was removed, and the cells were washed once with PBS. PBS wasremoved and replaced with a cell lysis buffer adopted from Laird et al.(Nucleic Acids Res. 19:4293, 1991). Briefly, 0.2 ml of lysis buffercontaining 200 mM NaCl, 100 mM Tris-HCl pH 8.5, 5 mM EDTA, 0.2% SDS, and100 ug/ml proteinase K was added to the well. The plate was returned tothe incubator for between three hours and overnight. The viscous celllysate was then transferred to a microfuge tube. An equal volume ofisopropanol was added to precipitate DNA. Following a 10 minute spin ina microfuge, the supernatant was discarded, and the pellet was washedonce with 0.5 ml of 70% ethanol. After removal of the ethanol, the DNApellet was air-dried and resuspended in 35 microliters of TE buffer (10mM Tris pH 8 and 1 mM EDTA). Aliquots of 3 μl were used for PCRanalysis.

PCR analysis DNA samples from drug resistant clones transduced with AAVparticles were screened for appropriate targeting of the vector usingPCR analysis. This screening strategy used one primer that annealswithin the DNA encoding the drug selection marker and another primerthat anneals within the targeted locus, but outside the sequence presentin the AAV targeting particles. PCR products are only detected if theAAV targeting DNA has integrated into the desired location of theendogenous genome.

Results from a single targeting experiment using these AAV particles areshown in FIG. 26. Based on this analysis, five out of 73 independentclones contained the appropriate targeted vector DNA.

This method may also be used with the AAV vector shown in FIG. 25 orwith any other appropriate adenovirus or adeno-associated viral vector.If desired, the second mu heavy chain allele can be mutated in theisolated colonies by transducing them with an AAV vector with adifferent antibiotic resistance gene (i.e., a gene other than a neomycinresistance gene). To select the resulting homozygous knockout cells, theinfected cells are cultured in the presence of the correspondingantibiotic. Alternatively, the isolated colonies can be transduced withan AAV vector containing a neomycin resistance gene and cultured in thepresence of a high concentration of antibiotic (i.e., a concentration ofantibiotic that kills heterozygous knockout cells but not homozygousknockout cells).

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

All publications mentioned in this specification are herein incorporatedby reference to the same extent as if each independent publication orpatent application was specifically and individually indicated to beincorporated by reference.

1. A transgenic bovine whose cells comprise one or more artificialchromosomes, each artificial chromosome comprising one or more humanimmunoglobulin heavy or light chain loci that undergo rearrangement andare expressed in B-cells to produce a human immunoglobulin in responseto exposure to one or more antigens.
 2. The transgenic bovine of claim1, wherein said one or more artificial chromosomes comprise a humanartificial chromosome.
 3. The transgenic bovine of claim 2, wherein saidhuman artificial chromosome is ΔHAC or ΔΔHAC.
 4. The transgenic bovineof claim 2, wherein said human artificial chromosome is derived from oneor more of human chromosome 14, human chromosome 2, and human chromosome22.
 5. The transgenic bovine of claim 1, wherein said immunoglobulinloci comprise both a human immunoglobulin light chain locus and humanimmunoglobulin heavy chain locus.
 6. An isolated B-cell obtained from atransgenic bovine of claim 1, wherein the B-cell comprises one or moreartificial chromosomes, each artificial chromosome comprising one ormore human immunoglobulin loci that undergo rearrangement and areexpressed to produce a human immunoglobulin in response to exposure toone or more antigens.
 7. An isolated transgenic bovine B-cell comprisingone or more artificial chromosomes, each artificial chromosomecomprising one or more human immunoglobulin heavy or light chain locithat undergo rearrangement and are expressed to produce a humanimmunoglobulin in response to exposure to one or more antigens.
 8. Thecell of claim 7, wherein said one or more artificial chromosomescomprise a human artificial chromosome.
 9. The cell of claim 8, whereinsaid human artificial chromosome is derived from one or more of humanchromosome 14, human chromosome 2, and human chromosome
 22. 10. The cellof claim 8, wherein said human artificial chromosome is ΔHAC or ΔΔHAC.11. The cell of claim 7, wherein said immunoglobulin loci comprise botha human immunoglobulin light chain locus and human immunoglobulin heavychain locus.
 12. A method of producing human antibodies, said methodcomprising the steps of: (a) administering one or more antigens ofinterest to a transgenic bovine whose cells comprise one or moreartificial chromosomes, each artificial chromosome comprising one ormore human immunoglobulin heavy or light chain loci that undergorearrangement and are expressed in B-cells, resulting in production ofhuman antibodies against said one or more antigens; and (b) recoveringsaid human antibodies from said bovine.
 13. The method of claim 12,wherein said one or more artificial chromosomes comprise a humanartificial chromosome.
 14. The method of claim 13, wherein said humanartificial chromosome is derived from one or more of human chromosome14, human chromosome 2, and human chromosome
 22. 15. The method of claim13, wherein said human artificial chromosome is ΔHAC or ΔΔHAC.
 16. Amethod of producing human antibodies to one or more antigens, saidmethod comprising recovering human antibodies from a transgenic bovinewhose cells comprise one or more artificial chromosomes, each artificialchromosome comprising one or more human immunoglobulin heavy or lightchain loci that undergo rearrangement and are expressed in B-cells,resulting in production of human antibodies against said one or moreantigens.
 17. The method of claim 16, wherein said one or moreartificial chromosomes comprise a human artificial chromosome.
 18. Themethod of claim 17, wherein said human artificial chromosome is derivedfrom one or more of human chromosome 14, human chromosome 2, and humanchromosome
 22. 19. The method of claim 17, wherein said human artificialchromosome is ΔHAC or ΔΔHAC.